Neuroprotective iron chelators and pharmaceutical compositions comprising them

ABSTRACT

Novel iron chelators exhibiting neuroprotective and good transport properties are useful in iron chelation therapy for treatment of a disease, disorder or condition associated with iron overload and oxidative stress, e.g., a neurodegenerative or cerebrovascular disease or disorder, a neoplastic disease, hemochromatosis, thalassemia, a cardiovascular disease, diabetes, an inflammatory disorder, anthracycline cardiotoxicity, a viral infection, a protozoal infection, a yeast infection, retarding aging, and prevention and/or treatment of skin aging and skin protection against sunlight and/or UV light. The iron chelator function is provided by a 8-hydroxyquinoline, a hydroxypyridinone or a hydroxamate moiety. The neuroprotective function is imparted to the compound, e.g., by a neuroprotective peptide. A combined antiapoptotic and neuroprotective function is provided by a propargyl group.

FIELD OF THE INVENTION

The present invention relates to amphiphilic metal chelators that havespecificity for iron and are useful for treatment of diseases, disordersand conditions by iron chelation therapy. In one aspect, the ironchelators are designed to provide desired cell membrane, particularlyblood-brain-barrier (BBB), transport properties in lipophilic media. Inanother aspect, these chelators are part of multifunctional compoundspossessing an iron chelator function and a residue that imparts aneuroprotective function and/or a residue that imparts bothantiapoptotic and neuroprotective functions. The invention furtherrelates to pharmaceutical compositions and methods for treatment and/orprevention of diseases, disorders and conditions associated withironoverload and oxidative stress, in particular neurodegenerative diseases,conditions and disorders such as Parkinson's disease, Alzheimer'sdisease, stroke, amyotrophic lateral sclerosis (ALS), multiplesclerosis, Friedreich's ataxia, Hallervorden-Spatz disease, epilepsy andneurotrauma.

BACKGROUND OF THE INVENTION

Iron is known to enhance the production of the highly reactive and toxichydroxyl radical, thus stimulating oxidative damage. Iron has beenassociated with a number of diseases, disorders and conditions becausehumans have no physiologic means of eliminating excess iron.

Hereditary hemochromatosis, a condition in which the body accumulatesexcess amounts of iron, is one of the most common genetic diseases inhumans. In the United States, as many as one million people haveevidence of hemochromatosis, and up to one in every ten people may carrythe gene for the disorder. Hemochromatosis is characterized by lifelongexcessive absorption of iron from the diet, with iron accumulating inbody organs, eventually causing inflammation and damage. Serious andeven fatal health effects can result, including cirrhosis of the liver,liver cancer, heart abnormalities (leading to heart failure), diabetes,impotence, and arthritis.

Clinical thalassemia (major and minor) are hereditary disorderscharacterized by defective production of hemoglobin, which leads todecreased production and increased destruction of red blood cells. Withsevere thalassemia, regular blood transfusions and folatesupplementation are given, resulting in iron overload. Since iron isusually not ingested in large amounts, the body holds onto what itreceives and has no way of ridding itself of any excess. Iron overloadis therefore the leading cause of death among thalassemia patients inindustrialized nations.

Patients with b-thalassemia major (TM) or refractory anemia (as inmyelodysplastic syndrome) who receive frequent or regular red-celltransfusions, coupled with increased iron absorption due to ineffectiveerytropoiesis, develop iron overload rapidly. The toxicity of ironbegins when its load exceeds the tissue or blood binding capacity tojoin a mobile intracellular or free nontransferrin-bound pool in theblood, the unbound iron accelerates hydroxyl radical formation resultingin peroxidative damage to cells. In the absence of chelation therapy,chronically transfused patients inevitably undergo progressivedeterioration in pancreatic, hepatic and cardiac function, and usuallysuccumb to life-threatening arrhythmias or intractable heart failure asa result of iron overload. This usually happens in the second decade oflife in poorly or unchelated patients with thalassemia major.

Deferoxamine (DFO), a naturally occurring siderophore, chelating iron ina labile intracellular pool that is itself rapidly renewed from thestorage pool, was introduced in early 1960s. The minimal absorption ofDFO from the gastrointestinal tract and its short half-life in the bloodnecessitate a slow prolonged parenteral administration of the drug toachieve net negative iron balance as the prime goal of an effectivechelation therapy. The expense and inconvenience of DFO has led to asearch for an orally-active iron chelator, and deferiprone (L1) has beenused in the last years for oral treatment of thalassemia major patients,but the risk of agranulocytosis mandates a careful evaluation of the useof this drug. Other orally active iron chelators have reached clinicaltrials in the past decade but their use in iron chelation therapy needmore investigation. The identification of suitable, preferably orally,effective iron chelators for the treatment of iron overload diseases,disorders and conditions still remains an unsolved problem.

Neurodegenerative diseases, such as Parkinson's disease (PD) andAlzheimer's disease (AD), are neurodegenerative syndromes for which atpresent no cure is available. Both diseases are the most widespreadneurodegenerative disorders. They affect approximately 0.5% and 4-8%,respectively, of the population over the age of 50 years, thereby,considering the still growing number of the elderly, forming anincreasing economic burden for society. Therefore, development of aneffective drug for preventing (neuroprotective) and treatingneurodegenerative diseases is essential for the whole society.

Numerous studies including in vivo, in vitro and relevant animal modelshave shown a linkage between free radical production andneurodegenerative diseases and disorders, such as Parkinson's diseases,Alzheimer's disease and stroke as well as ALS, multiple sclerosis,Friedreich's ataxia, Hallervorden-Spatz disease, epilepsy andneurotrauma.

For this reason, 8-hydroxyquinolines and hydroxypyridinones have beenproposed for iron binding as antioxidant-type drugs. Since ironaccumulation in neurodegenerative diseases is a common feature, theinventors have shown previously that it has a pivotal role in theprocess of neurodegenration (Youdim, 1988). We (Gassen and Youdim, 1999)and others (Cuajungco et al., 2000; Sayre et al., 2000) have suggestedon several occasions the development of iron chelators as therapeuticagents for Alzheimer's disease and Parkinson's disease.

In Parkinson's disease, the brain defensive mechanisms against theformation of oxygen free radicals are defective. In the substantia nigraof parkinsonian brains there are reductions in activities of antioxidantenzymes. Moreover, iron concentrations are significantly elevated inparkinsonian substantia nigra pars compacta and within the melanizeddopamine neurons. Latest studies have also shown that significantaccumulations of iron in white matter tracts and nuclei throughout thebrain precede the onset of neurodegeneration and movement disordersymptoms. Indeed the accumulation of iron at the site ofneurodegeneration is one of the mysteries of neurodegenerative diseasesbecause iron does not cross the BBB. Where the iron comes from and whyit accumulates is not known.

The etiology of Alzheimer's disease (AD) and the mechanism ofcholinergic neuron degeneration remain elusive. Nevertheless, thechemical pathology of AD shows many similarities to Parkinson's disease:the involvement of increased iron, release of cytochrome C, increasedalpha-synuclein aggregation, oxidative stress, loss of tissue reducedglutathione (GSH), an essential factor for removal of hydrogen peroxide,reduction in mitochondrial complex I activity, increased lipidperoxidation, and loss of calcium-binding protein 28-kDa calbindin, tomention a few. These similarities also include the progressive nature ofthe disease, proliferation of reactive microglia around and on top ofthe dying neurons, the onset of oxidative stress and inflammatoryprocesses.

Oxygen free radicals have been shown to be associated with proteindenaturation, enzyme inactivation, and DNA damage, resulting in lipidperoxidation of cell membranes, and finally the cell death inneurodegenerative diseases. One of the profound aspects ofneurodegenerative diseases is the accumulation and deposition ofsignificant amount of iron at the neurodegenerative sites. In AD, ironaccumulates within the microglia and within the neurons and in plaquesand tangles. Current reports have provided evidence that thepathogenesis of AD is linked to the characteristic neocorticalbeta-amyloid deposition, which may be mediated by abnormal interactionwith metals such as iron. Indeed, iron is thought to cause aggregationof not only beta-amyloid protein but also of alpha-synuclein, promotinga greater neurotoxicity. This has led to the notion that chelatable freeiron may have a pivotal role in the induction of the oxidative stressand the inflammatory process leading to apoptosis of neurons. Iron andradical oxygen species activate the proinflammatory transcriptionfactor, NFκB, which is thought to be responsible for promotion of thecytotoxic proinflammatory cytokines IL-1, IL-6 and TNF-alpha, whichincrease in AD brains is one feature of AD pathology. This is consideredlogical since iron, as a transition metal, participates in Fentonchemistry with hydrogen peroxide to generate the most reactive of allradical oxygen species, reactive hydroxyl radical. This radical has beenimplicated in the pathology of cell death and mechanism of action ofnumerous toxins and neurotoxins (6-hydroxydopamine, MPTP, kainite,streptocozin model of AD). Furthermore, such toxins mimic many of thepathologies of neurodegenerative diseases (AD, Parkinson's disease andHuntington's Chorea), one feature of which is the accumulation of iron,but not of other metals, at the site of neurodegeneration.

Iron alone or iron decompartmentalized from its binding site by aneurotoxin, e.g. the dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA),may induce oxidative stress and neurodegeneration, as evidenced inprevious studies of the inventors in which intranigral administration ofiron-induced “Parkinsonism” in rats and the iron chelatordesferrioxamine protected the rats against 6-OHDA-induced lesions ofnigrostrial dopamine neurons (Ben-Shachar et al., 1991). It has thusbeen suggested that treatment or retardation of the process ofdopaminergic neurodegeneration in the substantia nigra may be affectedby iron chelators capable of crossing the blood brain barrier in afashion similar to the copper chelator D-penacillamine used in thetreatment of Wilson's disease. This therapeutic approach for thetreatment of Parkinson's disease can be applied to othermetal-associated neurological disorders such as tardive dyskinesia,Alzheimer's and Hallervorden-Spatz diseases.

Stroke is the third leading cause of death in the Western world today,exceeded only by heart diseases and cancer. The overall prevalence ofthe disease is 0.5-0.8% of the population. Stroke is characterized by asudden appearance of neurological disorders such as paralysis of limbs,speech and memory disorders, sight and hearing defects, etc., whichresult from a cerebrovascular damage.

Haemorrhage and ischemia are the two major causes of stroke. Theimpairment of normal blood supply to the brain is associated with arapid damage to normal cell metabolism including impaired respirationand energy metabolism lactacidosis, impaired cellular calciumhomeostasis release of excitatory neurotransmitters, elevated oxidativestress, formation of free radicals, etc. Ultimately these events lead tocerebral cell death and neurological disfunction.

Treatment of stroke is primarily surgical. Much effort is being investedin less aggressive therapeutical intervention in the search for drugswhich are capable of restoring normal blood perfusion in the damagedarea as well as drugs which are designed to overcome the above listeddamaging events associated with cellular damage.

Oxidative stress and free radical formation play a major role in tissueinjury and cell death. These processes are catalyzed by transient metalions, mainly iron and copper. In the case of stroke, since vasculardamage is involved, iron is available for the free radical formation, aprocess that could be prevented by iron chelators. Indeed, withlazaroides (21-amino steroids), known free radical scavengers, asignificant improvement of local and global ischemia damages induced inanimals has been achieved.

Iron chelators and radical scavengers have been shown to have potentneuroprotective activity in animal models of neurodegeneration. However,the major problem with such compounds is that they do not cross the BBB.The prototype iron chelator Desferal (desferrioxamine) was first shownby us to be a highly potent neuroprotective agent in animal models ofParkinson's disease (Ben-Schachar et al., 1991). However, Desferal doesnot cross the BBB and has to be injected centrally. Desferal alsoprotects against streptozocin model of diabetes.

Free radicals in living organism are believed to be produced by thereaction of transition metal ions (especially copper and iron) withpoorly reactive species such as H₂O₂, [O₂.⁻], thiols, lipid peroxides.Antioxidant metal chelators, by binding free metal ions (especiallycopper and iron) or metal ions from active centers of enzymes of thedefense system, can influence the oxidant/antioxidant balance in vivo,and hence, may affect the process of dopaminergic and cholinergicneurodegeneration and have great therapeutic potential againstneuodegenerative diseases.

Iron accumulation in aging and the resulting oxidative stress has beensuggested to be a potential causal factor in aging and age-relatedneurodegenerative disorders (Butterfield et al., 2001). There isincreasing evidence that reactive oxygen species play a pivotal role inthe process of ageing and the skin, as the outermost barrier of thebody, is exposed to various exogenous sources of oxidative stress, inparticular UV-irradiation. These are believed to be responsible for theextrinsic type of skin ageing, termed photo-ageing. (Podda et al.,2001). Iron chelators have thus been suggested to favor successfulageing in general, and when applied topically, successful skin ageing(Polla et al., 2003).

Iron is a factor in skin photodamage, not only in ageing, apparently byway of its participation in oxygen radical production. Certain topicaliron chelators were found to be photoprotective (Bisset and McBride,1996; Kitazawa et al., 1999). UVA radiation-induced oxidative damage tolipids and proteins in skin fibroblasts was shown to be dependent oniron and singlet oxygen (Vile and Tyrrel, 1995). Iron chelators can thusbe used in cosmetic and non-cosmetic formulations, optionally withsunscreen compositions, to provide protection against UV radiationexposure.

Other diseases, disorders or conditions associated with iron overloadinclude: (i) viral infections, including HIV infection andAIDS—oxidative stress and iron have been described to be important inthe activation of HIV-1 and iron chelation, in combination withantivirals, might add to improve the treatment of viral, particularlyHIV disease (van Asbeck et al., 2001); (ii) protozoal, e.g. malaria,infections; (iii) yeast, e.g. Candida albicans, infections; (iv)cancer—several iron chelators have been shown to exhibit anti-tumoractivity and may be used for cancer therapy either alone or incombination with other anti-cancer therapies (Buss et al., 2003); (v)iron chelators may prevent cardiotoxicity induced by anthracyclineneoplastic drugs (Hershko et al., 1996); (vi) inflammatorydisorders—iron and oxidative stress have been shown to be associatedwith inflammatory joint diseases such as rheumatoid arthritis (Andrewset al., 1987; Hewitt et al., 1989; Ostrakhovitch et al., 2001); (vii)diabetes—iron chelators have been shown to delay diabetes in diabeticmodel rats (Roza et al., 1994); (viii) iron chelators have beendescribed to be potential candidates for treatment of cardiovasculardiseases, e.g. to prevent the damage associated with free radicalgeneration in reperfusion injury (Hershko, 1994; Flaherty et al., 1991);(ix) iron chelators may be useful ex-vivo for preservation of organsintended for transplantation such as heart, lung or kidney (Hershko,1994).

One of the main problems in the use of chelating agents asantioxidant-type drugs is the limited transport of these ligands ortheir metal complexes through cell membranes or other biologicalbarriers.

Drugs with the brain as the site of action should, in general, be ableto cross the blood brain barrier (BBB) in order to attain maximal invivo biological activity. The efficacy of the best establishediron-chelating drug, Desferal, in the neurodegenerative diseases, islimited by its ineffective transport property and high cerebro- andoculotoxicity.

8-Hydroxyquinoline is a strong chelating agent for iron, and containstwo aromatic rings, which can scavenge free radicals by themselves. Inour previous PCT Publication No. WO 00/74664, various iron chelatorshave been disclosed and their action in Parkinson's disease preventionhas been shown. The lead compound,5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline (hereinreferred to as VK-28), was able to cross the BBB and was shown to beactive against 6-hydroxydopamine (6-OHDA) in an animal model ofParkinson's disease.

It would be very desirable to provide novel iron chelators that exhibitalso neuroprotective activity and/or good transport properties throughcell membranes including the blood brain barrier.

SUMMARY OF THE INVENTION

The present invention relates to amphiphilic metal chelators that havespecificity for iron and exhibit neuroprotective and/or good transportproperties in lipophilic media.

In one aspect, the invention provides a compound comprising an ironchelator function and a residue selected from the group consisting of aresidue that imparts a neuroprotective function to the compound, aresidue that imparts combined antiapoptotic and neuroprotectivefunctions to the compound, or both, and pharmaceutically acceptablesalts thereof.

The iron chelator function is provided preferably by a8-hydroxyquinoline residue, a hydroxamate residue, or a pyridinoneresidue, the neuroprotective function may be provided by a cysteine oralanine residue or by the residue of a neuroprotective peptide, aneuroprotective analog or a neuroprotective fragment thereof, and thecombined antiapoptotic and neuroprotective functions is preferablyprovided by a propargyl group.

In another aspect, the invention provides compounds of the formulas I toIV as defined hereinafter in the description and in the claims, andpharmaceutically acceptable salts thereof.

The compounds of the present invention are useful for treatment and/orprevention of diseases, disorders and conditions associated with ironoverload and oxidative stress such as, but not limited to,neurodegenerative and cerebrovascular diseases and disorders, neoplasticdiseases, hemochromatosis, thalassemia, cardiovascular diseases,diabetes, inflammatory disorders, anthracycline cardiotoxicity, viral,protozoal and yeast infections, for retarding ageing, and for preventionand/or treatment of skin ageing and/or skin damage associated with skinageing and/or exposure to sunlight and/or UV light.

In a further aspect, the present invention provides a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and acompound of the invention.

In yet another aspect, the present invention provides a cosmeticcomposition comprising a compound of the invention.

In still a further aspect, the present invention provides the use of acompound of the invention for the preparation of a pharmaceuticalcomposition for iron chelation therapy.

In still another aspect, the present invention provides a method foriron chelation therapy which comprises administering to an individual inneed thereof an effective amount of a compound of the invention or of apharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF THE FIGURES

The formulas of the compounds defined by the adopted designation used inthe description of the specification and in the claims appear in theAppendices I to VI at the end of the description, just before theclaims.

FIGS. 1A-1C are graphs showing iron chelation (in solution) by thechelators of the invention HLM7, HLM8, HLM9, HLA16, HLA20 (FIG. 1A),HLA16, HLA20, M9, M10 (FIG. 1B), and M7, M11, M12 (FIG. 1C), and theknown iron chelators deferiprone (L1) and VK-28, using iron precomplexedcalcein (Fe-CAL) solutions.

FIGS. 2A-2D show iron permeation into K562 cells of the chelators of theinvention HLM7, HLM8, HLM9, HLA16, HLA20 (FIG. 2A), HLA16, HLA20, M9,M10 (FIG. 2B), and M7, M11, M12 (FIG. 2C), HLM9, HLA16, HLA20 (FIG. 2D),and the known iron chelators deferiprone (L1), VK-28 desferrioxamine B(DFO), and salicylaldehyde isonicotinoyl hydrazone (SIH).

FIG. 3 shows neuroprotective effect of the chelators of the inventionHLM7, HLM8, HLM9, HLA16, M7, HLA20 and of apomorphine (Apo) ondifferentiated neuronal P19 cells treated with 6-OH-dopamine.

FIGS. 4A-4B show protection by of PC12 cells against serumdeprivation-induced apoptosis by the chelators of the invention M32(FIG. 4A) and HLA20 (FIG. 4B), in comparison to the known chelators Rasaline and VK-28.

FIGS. 5A-5D are graphs showing the in vitro inhibitory action of HLA20and M32 (FIG. 5A), M30 and M31 (FIG. 5B), VK-28, and propargylamine (P)against rat brain MAO-B, and of M31 (FIG. 5C), M32 and HLA20 (FIG. 5D),VK-28, and propargylamine (P) against rat brain MAO-B and MAO-A.

FIG. 6 shows inhibition of lipid peroxidation by the chelators of theinvention HLM7, HLM8, HLM9, HLA16 and HLA20 in the presence of 5.0 μMFe₂SO₄/50 μM ascorbic acid. The results are the mean±SEM, n=3, p<0.05.

DETAILED DESCRIPTION OF THE INVENTION

In one broad aspect, the present invention provides a compoundcomprising iron chelator function and a residue selected from the groupconsisting of a residue that imparts a neuroprotective function to thecompound, a residue that imparts combined antiapoptotic andneuroprotective functions to the compound, or both.

In one most preferred embodiment, the residue imparting combinedantiapoptotic and neuroprotective functions is a propargyl moiety,described recently as playing a crucial role in the antiapoptotie andneuroprotective effects of anti-Parkinson drug rasagiline[N-propargyl-(1R)-aminoindan] (Yogev-Falach et al., 2003).

The iron chelator function in the compounds of the invention is providedby a residue selected from the group consisting of a 8-hydroxyquinolineresidue, a hydroxamate residue, and a pyridinone residue.

In one preferred embodiment, the iron chelator function is provided by a8-hydroxyquinoline group to which the residue imparting theneuroprotective function and/or combined antiapoptotic andneuroprotective functions may be linked through the 5, 6 or 7 positionof the quinoline ring. In a more preferred embodiment, theiron-chelating group is the 8-hydroxy-5-quinolinyl group of the formulabelow and, most preferably, it is a 8-hydroxy-5-quinolinylmethylenegroup.

In another preferred embodiment, the iron chelator function is providedby or a 3-hydroxypyridin-4-one or 1-hydroxypyridin-2-one of the formulasbelow:

In the formulas above, R represents the group carrying theneuroprotective function and/or combined neuroprotective andantiapoptotic functions that may be linked at position 5, 6 or 7 of thequinoline ring, at position 1, 2, 5 or 6 of the 3-hydroxy-4-pyridinonering that may be further substituted by a lower alkyl, preferably,methyl group, and at position 4 or 5 of the 1-hydroxy-2-pyridinone ring.In one preferred embodiment, the iron-chelating function is provided bya 2-methyl-3-hydroxy-4-pyridinone group substituted at position 1 by thedesired further functions.

The proposed N-hydroxypyridin-2-ones and 3-hydroxypyridin-4-ones are asuitable type of candidate iron chelators for three reasons: (1)hydroxypyridinones are the most promising oral iron chelatorsconsidering both their properties and the results of biologicaltrials—one hydroxypyridinone, deferiprone (CP20 or L1), is an orallyiron chelating agent which is used worldwide in thalassaemia, cancer,leukaemia, haemodialysis and other patients (Kontoghiorghes, 2001); (2)some hydroxypyridinones, for example CP20, CP24, CP94, have been provedto be able to cross the BBB (Crivori et al., 2000); and (3) there aremany structural similarities between hydroxypyridinones with catechol,an iron chelator group in the DOPA structure.

In another embodiment, the iron-chelating function is provided by ahydroxamate group. Hydroxamtes are known as iron chelators. For decades,desferrioxamine B (Desferal) has been the therapeutic iron chelator ofchoice for iron-overload treatment, despite numerous problems associatedwith its use.

In one embodiment of the invention, the residue imparting aneuroprotective function to the compound of the invention is selectedfrom the group consisting of a neuroprotective peptide, aneuroprotective analog and a neuroprotective fragment thereof.

The neuroprotective peptide that can be used in the compounds of theinvention may be, without being limited to, vasoactive intestinalpeptide (VIP), gonadotropin-releasing hormone (GnRH), Substance P andenkephalin.

In one preferred embodiment, the neuroprotective function is provided bythe residue of VIP, GnRH, Substance P car enkephalin or a fragmentthereof in which one amino acid residue is replaced by a L- orD-cysteine residue, to which the iron-chelating residue is linked viathe —S— atom of the L- or D-Cys residue.

one embodiment of the invention, the neuroprotective peptide vasoactiveintestinal peptide (VIP), a basic 28 amino acid peptide, origin isolatedfrom the gastrointestinal system, that has been shown to protect neuronsthe central nervous system from a variety of neurotoxic substancesincluding the envelop protein from HIV, tetrodotoxin, and the betaamyloid peptide. The beta amyloid peptide is a major component of thecerebral, amyloid plaque in Alzheimer disease and has been shown to beneurotoxic and associated with Alzheimer disease onset and progression,VIP itself presents a limitation for clinical use as a possibleneuroprotectant since it does not cross the BBB. Lipophilic derivativesof VIP, such as stearyl-VIP, do cross the BBB. However, for moreefficient penetration and as well as from economical aspects, it isdesirable to have much smaller molecules mimicking the parent peptide(VIP) activity while possessing better penetration through biologicalbarriers. In the laboratory of one of the present inventors it was foundthat a 4-amino acids lipidic derivative of VIP(stearyl-Lys-Lys-Tyr-Leu-NH₂—SEQ ID NO:1) surpasses the activity of theparent peptide (VIP), through binding and activation of its receptor,and provides a lead compound for drug design against neurodegenerativediseases (Gozes et al., 1999).

Thus, in one embodiment of the invention, the neuroprotective functionis imparted by a VIP fragment of the SEQ ID NO: 2:

Z-Lys-Lys-Cys-Leu-NH₂

wherein Z is H or a hydrophobic group such as stearyl (St) or Fmoc.

In one embodiment, the iron chelator is a 8-hydroxyquinolinylmethylgroup and the compound has the formula:

In this model, the peptide Lys-Lys-Tyr-Leu-NH₂ (SEQ ID NO:1) is the coreactive site for neuroprotective effect. The aromatic phenolic moiety ofTyr is substituted by an iron chelator 8-hydroxyquinoline, which servesas an antioxidative active core. The hydrophobic group R may be used toadjust the lipophilicity of the whole molecule so as to control thetransport properties through the BBB.

Examples of compounds containing the above formula containing a8-hydroxy-5-quinolinylmethyl residue (HQ) are the compounds hereindesignated StKKC(HQ)L (M6) and Fmoc-KKC(HQ)L (M7) in Appendix II. Thecorresponding compounds herein designated StKKC(HQ-Pr)L (M6A) andFmoc-KKC(HQ-Pr)L (M7A) in Appendix I have in addition a propargylamino(Pr) group linked to the —CH₂ group at the 5-position of the quinolinering.

Examples of further compounds of the invention comprising a VIP fragmentanalog of SEQ ID NO: 2 are the hydroxamates herein designated M6B andM7B in Appendix V that comprise also an aminopropargyl group.

Gonadotropin-releasing hormone (GnRH) is a neurohormone produced in thehypothalamic neurosecretory cells that controls release of thegonadotropins luteinizing hormone (LH) and follicle-stimulating hormone(FSH) from the anterior pituitary. The peptide was later found tofunction in the brain as a neurotransmitter and/or neuromodulator and tohave good transport properties (GnRH is able to cross the BBB). SeveralGnRH analogs are now either in clinical trials or in clinical use forcontraception and for the treatment of various hormone-dependentdiseases including prostate and breast cancers. The combination of GnRHanalogs with an iron chelator, e.g. hydroxamate, pyridinone or8-hydroxyquinoline as an antioxidative core, is envisaged specially asprotective agents in the treatment of neurodegenerative diseases.

GnRH is a decapeptide of the sequence below (the 5-oxo proline at theamino terminal is sometimes presented as pyroglutamic acid—pGlu):

(SEQ ID NO: 3) 5-oxo-Pro-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂

Two analogs of GnRH were prepared in which the amino acid residue atposition 5 (Tyr) or 6 (Gly) was replaced by L-Cys or D-Cys,respectively. These modifications are not expected to significantlyaffect the bioactivity of GnRH. Moreover these changes may generatesuperagonists. The free SH group of this analogs at the cysteine (5 or6) position makes these compounds good candidates for specific chemicalmodifications. This change, ie, at position 6, also serves to improvethe stability of the analog to proteolysis.

The resulting GnRG analogs have the sequences identified by SEQ ID NO:4and SEQ ID NO:5, as follows:

(SEQ ID NO: 4) 5-oxo-Pro-His-Trp-Ser-Cys-Gly-Leu-Arg-Pro-Gly-NH₂(SEQ ID NO: 5) 5-oxo-Pro-His-Trp-Ser-Tyr-D-Cys-Leu-Arg-Pro-G1y-NH₂

To the S atom of the L-Cys or D-Cys residue the iron chelator functionis attached. For example, the resulting compounds with a8-hydroxy-5-quinolinylmethyl residue (HQ) are the compounds hereindesignated L-Cys⁵(HQ)GnRH (M8) and D-Cys⁶(HQ)GnRH (M22) in Appendix II.The corresponding compounds herein designated L-Cys⁵(HQ-Pr)GnRH (M8) andD-Cys⁶(HQ-Pr)GnRH (M22) in Appendix I have in addition a propargylamino(Pr) group linked to the —CH₂ group at the 5-position of the quinolinering.

Examples of further compounds of the invention comprising a GnRH analogof SEQ ID NO:4 or SEQ ID NO:5 are the hydroxamates herein designated M8Band M22B in Appendix V that comprise also a propargylamino group.

According to the invention, similar Cys-containing GnRH analogs can beprepared starting from analogs of GnRH such as, but not limited to,leuprolide, nafarelin, goserelin, histrelin, and D-Lys⁶GnRH, and thenattaching them to a HQ, pyridinone or hydroxamate residue and,optionally, to a propargylamino residue.

Substance P (SP) is a peptide neurotransmitter widely distributed in theperipheral and central nervous systems of vertebrates. SP is involved inmodulating neuronal nicotinic acetylcholine receptors (nAChRs) in thesympathetic nervous system. Substance P analogs may be useful as drugsin control of neurodegenerative diseases.

Substance P is a undecapeptide of the sequence:

(SEQ ID NO: 6) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂

Two analogs of Substance P were prepared in which the amino acid residueat position 7 (Phe) or 8 (Phe) was replaced by Cys, resulting in thepeptides of the sequences:

(SEQ ID NO: 7) Arg-Pro-Lys-Pro-Gln-Gln-Cys-Phe-Gly-Leu-Met-NH₂(SEQ ID NO: 8) Arg-Pro-Lys-Pro-Gln-Gln-Phe-Cys-Gly-Leu-Met-NH₂

To the S atom of the Cys residue the iron chelator function is attached.For example, the resulting compounds with a 8-hydroxy-5-quinolinylmethylresidue (HQ) are the compounds herein designated Cys⁷(HQ)Substance-P(M27) and Cys⁸(HQ) Substance-P (M28) in Appendix II. The correspondingcompounds herein designated Cys⁷(HQ-Pr)Substance-P (M27A) andCys⁸(HQ-Pr) Substance-P (M28A) in Appendix I have in addition apropargylamino group (Pr) linked to the —CH₂ group at the 5-position ofthe quinoline ring.

Examples of further compounds of the invention comprising a Substance-Panalog of SEQ ID NO:7 or SEQ ID NO:8 are the hydroxamates hereindesignated M27B and M28B in Appendix V that comprise also apropargylamino group.

Met⁵-enkephalin and Leu⁵-enkephalin are two naturally occurringpentapeptides belonging to the endorphin class, of the sequences,respectively:

Tyr-Gly-Gly-Phe-Met (SEQ ID NO: 9) Tyr-Gly-Gly-Phe-Leu. (SEQ ID NO: 10)

Two analogs of each of Met⁵-enkephalin (SEQ ID NO:9) and Leu⁵-enkephalin(SEQ ID NO:10) have been prepared in which either the Phe residue atposition 4 or the Tyr residue at position 1 was replaced by a Cysresidue, as follows:

Tyr-Gly-Gly-Cys-Met (SEQ ID NO: 11) Cys-Gly-Gly-Phe-Met (SEQ ID NO: 12)Tyr-Gly-Gly-Cys-Leut (SEQ ID NO: 13) Cys-Gly-Gly-Phe-Leu (SEQ ID NO: 14)

To the S atom of the Cys residue the iron chelator function is attached.For example, the resulting compounds with a 8-hydroxy-5-quinolinylmethylresidue (HQ) are the compounds herein designated YGGC(HQ)L (M18) for SEQID NO: 13, YGGC(HQ)M (M19) for SEQ ID NO: 11, C(HQ)GGFL (M20) for SEQ IDNO: 14, and C(HQ)GGFM (M21) for SEQ ID NO: 12, in Appendix II. Thecorresponding compounds herein designated YGGC(HQ-Pr)L (M18A),YGGC(HQ-Pr)M (M19A), C(HQ-Pr)GGFL (M20A), and C(HQ-Pr)GGFM (M21A) inAppendix I have in addition a propargylamino (Pr) group linked to the—CH₂ group at the 5-position of the quinoline ring.

Examples of further compounds of the invention comprising an enkephalinanalog of SEQ ID NO:11-14 are the hydroxamates herein designated M18BA,M19B, M20B and M21B in Appendix V that comprise also a propargylaminogroup.

In another embodiment of the invention, the residue imparting aneuroprotective function to the compound of the invention is selectedfrom the group consisting of a L or D cysteine or alanine residue.

When the neuroprotective function is L- or D-cysteine, the iron chelatorfunction is attached to the S atom of the Cys residue. For example, theresulting compounds with a 8-hydroxy-5-quinolinylmethyl residue (HQ) arethe compounds herein designated D-HQ-CysOH (M11) and L-HQ-CysOH (M12) inAppendix II, and the corresponding compounds herein designatedD-(HQ-Pr)-CysOH (M11a) and L-(HQ-Pr)-CysOH (M12a) in Appendix III.Examples of further compounds of the invention comprising a cysteineresidue are the hydropyridinone derivatives herein designated M11b andM12b in Appendix VI that contain a propargylamino group.

Examples of compounds of the invention wherein the neuroprotectivefunction is L- or D-alanine are the compounds with a8-hydroxy-5-quinolinyl residue (HQ) herein designated D-HQ-Ala (M9),L-HQ-Ala (M10), the internal salt HQAla (HLM8), and the ethyl esterHQAlaEt (HLM9) in Appendix IV, and the corresponding compounds hereindesignated D-(HQ-Pr)-Ala (M9a), L-(HQ-Pr)-Ala (M10a), in Appendix III. Afurther example is the hydroxypyridinone derivative herein designatedM9b in Appendix VI.

Parkinson's disease is associated with decreased dopaminergic functionin the brain, and many of the symptoms of the disease can be alleviatedby oral administration of L-DOPA (L-dihydroxyphenylalanine). L-DOPA isconverted to dopamine by DOPA decarboxylase in the brain. Based onretrosynthesis analysis of the lead compound DOPA, which contains ironchelating group (catechol) and an amino acid (alanine), theiron-chelator-peptide and iron chelator-amino acid derivatives describedabove may be useful drug candidates as antioxidant-type drugsconstituting another class of antiparkinsonism, and also potentiallyother neurodegenerative diseases and other disorders in which ironchelators are useful.

As mentioned before, drugs with the brain as the site of action should,in general, be able to cross the blood brain barrier (BBB) in order toattain maximal in vivo biological activity. When the intention is tobring to the brain iron chelators to bind and remove iron thataccumulates in the brain in some neurodegenerative diseases, one of thepossible solutions is to design iron-chelating molecules with specificgroups, responsible for amphiphilic behavior. Such amphiphilic groupspossess lipophilic and hydrophilic centers. The size and structure ofboth centers control the overall lipophilicity of the whole molecule,and hence its transport properties.

The present invention further provides amphiphilic metal chelators thathave specificity for iron and are designed to provide desired bloodbrain barrier (BBB) transport properties in lipophilic media.

In one embodiment, the invention relates to compounds comprising an ironchelating residue that crosses the BBB wherein said iron chelatingresidue is a 8-hydroxy-5-quinolinylmethyl group linked to an aliphaticchain via a linker selected from the group consisting of anethylenediamine moiety or a piperazine or 1,3,5-perhydrotriazine ring asshown below, but excluding the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxy-quinoline.

According to this embodiment, the 8-hydroxyquinoline part of themolecule serves both as iron-specific chelator and radical-scavenger andR is an aliphatic

chain of different lengths, but is not a 2-hydroxyethyl group when thelinker is a piperazine ring. By changing R, we can adjust the size andlipophilicity of the whole molecule, thus controlling the transportproperties through the BBB. In a preferred embodiment, the linker is apiperazine ring.

The above iron chelators when the linker is piperazine may be preparedby reacting piperazine with di(t-butyl)dicarbonate (Boc₂O), followed byreaction of the Boc-protected piperazine with a halide R—X (R is apropargyl containing group, X is hal), removal of the Boc group withTFA, and reaction with 8-hydroxy-5-chloromethylquinoline, as describedin the scheme below:

The lipid solubility of the whole molecule, which is a key factor incontrolling the transport property of a drug through the BBB, can beadjusted by changing n. The aliphatic chain may also contain aheteroatom selected from O, S and N. Examples of such compoundsaccording to the invention are the compounds herein designated HLA16a,HLA20 and M17 in Appendix III. Also envisaged by the invention isCompound HLA16 in Appendix IV, that has no propargyl group.

The present invention encompasses compounds of the formulas I to IV:

wherein

R₁ is a residue of an analog of a neuroprotective peptide or a fragmentthereof containing a cysteine residue that is linked to the C atom viathe —S— atom of the Cys residue, and wherein the amino terminal of thepeptide is optionally substituted by a hydrophobic group such as Fmoc orstearyl;

R₂ is H or —NH—X;

R₃ is a group selected from the group consisting of

-   -   (i) —NH—CH₂—CH₂—NH—R₄;

-   -   (ii) —CR₅R₆R₇; —N(CH₃)—X; (iv) —N(R₈)—CH(CH₂SH)COOC₂H₅;    -   (v) —N(R₈)—CH₂—COOCH₂C₆H₅; and (vi) —S—CH₂—CH(COOH)—NHR₈′;

R₄ is selected from the group consisting of (i) X; (ii) COOC₂H₅;

-   -   (iii) (CH₂)₂—O—R₈; and (iv) —COO—(CH₂)₂—NH—R₈;

R₅ is H, C₁-C₄ lower alkyl, preferably CH₃, or COOC₂H₅;

R₆ is H, COOH, COO⁻ or COOC₂H₅;

R₇ is selected from the group consisting of (i) —NH—R₈; (ii) —NH₃ ⁺;

-   -   (iii) —NH—COCH₃; (iv) —NH—NH—R₈;    -   and (v) —NH—NH—CO—CH(CH₂OH)—NH—R₃;

R₈ is H or X, and R′₈ is H, X, or Fmoc;

R₉ is selected from the group consisting of (i) H; (ii) —CO—CH₂—R₁;

-   -   (iii) —CH₂—COOCH₂C₆H₅; (iv) —CH(CH₂SH)COOC₂H₅;

R₁₀ is X; —CH₂—CH(SH)COOC₂H₅; or

n is an integer from 1 to 6;

R₁₁ is a group selected from the group consisting of

(i) —S—CH₂—CH(COOH)—NH—X;

(ii) —N(X)—CH₂COO—CH₂—C₆H₅;

(iii) —N(CH₃)—X;

(iv) (iv) —N(X)—CH(CH₂SH)COOC₂H₅;

(v) —CH₂—NH—NH—CO—CH(CH₂OH)—NH—X;

(vi) —C(CH₃)(COOH)—NH—NH—X,

(vii) —CH(COOH)—NH—X;

(viii) —CH(COOC₂H₅)—NH—X; and

R₁₂ is X, C₁-C₄ lower alkyl, preferably methyl, COOC₂H₅, or —(CH₂)₂—OH;

R₁₃ is X, —(CH₂)₂—OX, or —COO—(CH₂)₂—NH—X; and

X is a propargyl group,

but excluding the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxy-quinoline.

In one preferred embodiment, the compound of the invention is a compoundof the formula I above

R₁ is a residue of an analog of a neuroprotective peptide or a fragmentthereof containing a cysteine residue that is linked to the C atom viathe —S— atom of the Cys residue, and wherein the amino terminal of thepeptide is optionally substituted by a hydrophobic group such as Fmoc orstearyl;

R₂ is H or —NH—X; and

X is a propargyl group.

R₁ is preferably the residue of an analog of VIP, GnRH, Substance P orenkephalin or a fragment thereof in which one amino acid residue isreplaced by a cysteine residue, more preferably the VIP fragment analogsof SEQ ID NO:2 that may bear a stearyl or a Fmoc group at the aminoterminal, the GnRH analogs of SEQ ID NO:4 and SEQ ID NO:5, the SubstanceP analogs of SEQ ID NO:7 and SEQ ID NO:8, and the enkephalin analogs SEQID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

In preferred embodiments, the compounds of formula I are the compoundswherein R₂ is H and R₁ is selected from the group consisting of theresidue of a VIP fragment analog of SEQ ID NO:2 bearing a stearyl (M6,Appendix II) or a Fmoc group (M7, Appendix II) at the amino terminal,the residue of a GnRH analog of SEQ ID NO:4 (M8, Appendix II) or SEQ IDNO:5 (M22, Appendix II), the residue of a Substance P analog of SEQ IDNO:7 (M27, Appendix II) or SEQ ID NO:8 (M28, Appendix II), and theresidue of an enkephalin analog of SEQ ID NO:11 (M19, Appendix II), SEQID NO:12 (M21, Appendix II), SEQ ID NO:13 (M18, Appendix II), and SEQ IDNO:14 (M20, Appendix II).

In more preferred embodiments, the compounds of formula I are thecompounds wherein R₂ is a propargylamino group and R₁ is selected fromthe group consisting of the residue of a VIP fragment analog of SEQ IDNO:2 bearing a stearyl (M6A, Appendix I) or a Fmoc group (M7A, AppendixI) at the amino terminal, the residue of a GnRH analog of SEQ ID NO:4(M8A) or SEQ ID NO:5 (M22A, Appendix I), the residue of a Substance Panalog of SEQ ID NO:7 (M27A, Appendix I) or SEQ ID NO:8 (M28A, AppendixI), and the residue of an enkephalin analog of SEQ ID NO:11 (M19A,Appendix I), SEQ ID NO:12 (M21A, Appendix I), SEQ ID NO:13 (M18A,Appendix I), and SEQ ID NO:14 (M20A, Appendix I).

In another preferred embodiment of the invention, there is provided acompound of formula II:

-   -   R₃ is a group selected from the group consisting of    -   (i) —NH—CH₂—CH₂—NH—R₄;

-   -   (ii) —CR₅R₆R₇; (iii) —N(CH₃)—X; (iv) —N(R₈)—CH(CH₂SH)COOC₂H₅;    -   (v) —N(R₈)—CH₂—COOCH₂C₆H₅; and (vi) —S—CH₂—CH(COOH)—NHR₈′;

R₄ is selected from the group consisting of (i) X; (ii) COOC₂H₅;

-   -   (iii) (CH₂)₂—O—R₈; and (iv) —COO—(CH₂)₂—NH—R₈;

R₅ is H, CH₃ or COOC₂H₅;

R₆ is H, COOH, COO⁻ or COOC₂H₅;

R₇ is selected from the group consisting of (i) —NH—R₈; (ii) —NH₃ ⁺;

-   -   (iii) —NH—COCH₃; (iv) —NH—NH—R₈;    -   and (v) —NH—NH—CO—CH(CH₂OH)—NH—R₈;

R₈ is H or X, and R′₈ is H, X, or Fmoc; and

X is a propargyl group,

but excluding the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxy-quinoline.

In a more preferred embodiment, in the compound of formula II, R₃ is apiperazine ring to which an aliphatic chain R₄ is linked to the nitrogenatom at the 4-position, but excluding the compound wherein R₄ is—(CH₂)₂—OH. This compound,5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxy-quinoline, isherein identified in Appendix IV as VK-28, and is known from WO00/74664. In one preferred embodiment, the8-hydroxy-5-quinolinylmethylpiperazine compounds of the invention do notcontain a propargyl group (R₄ is —COOC₂H₅ or R₈ is H), as represented bythe compound herein designated HLA16 (Appendix IV). In another preferredembodiment, the 8-hydroxy-5-quinolinylmethylpiperazine compounds of theinvention contain a propargyl group (R₈ is X), as represented by thecompounds herein designated HLA16a, HLA20, and M17 (Appendix

In another more preferred embodiment, in the compound of formula II, R₃is —S—CH₂—CH(COOH)—NHR₈′, wherein R₈′ is H, namely, R₃ is the residue ofL-Cys or D-Cys, as represented by the compounds herein designatedD-HQ-CysOH (M11, Appendix II) and L-HQ-CysOH (M12, Appendix II), or R₈′is propargyl, as represented by the compounds herein designatedD-(HQ-Pr)-CysOH (M11a, Appendix III) and L-(HQ-Pr)-CysOH (M12a, AppendixIII), or R₈′ is Fmoc, as represented by the compounds herein designatedM11B and m12B (Appendix IV).

In another more preferred embodiment, in the compound of formula II, R₃is a group —CR₅R₆R₇, wherein R₅ is H, R₆ is COOH, and R₇ is —NH—R₈,wherein R₈ is H, namely, R₃ is the residue of L-Ala or D-Ala, asrepresented by the compounds herein designated D-HQ-Ala (M9, AppendixIV) and L-HQ-Ala (M10, Appendix IV); or R₈ is propargyl, as representedby the compounds herein designated D-(HQ-Pr)-Ala (M9a, Appendix III) andL-(HQ-Pr)-Ala (M10a, Appendix III); or R₅ is H, R₆ is COO⁻ and R₇ is—NH₃ ⁺, as represented by the compound herein designated HQ-Ala (HLM8,Appendix IV); or R₅ is H, R₆ is COOC₂H₅ and R₇ is —NH₂, as representedby the compound herein designated HQ-AlaEt (HLM9, Appendix IV); or R₅and R₆ are both COOC₂H₅, and R₇ is —NH—COCH₃, as represented by thecompound herein designated HLM7 (Appendix IV); or R₅ is H, R₆ is COOC₂H₅and R₇ is —NH-propargyl, as represented by the compound hereindesignated M3I (Appendix III).

In another more preferred embodiment, in the compound of formula II, R₃is a group —NR₈—CH(CH₂SH)COOC₂H₅, wherein R₈ is H or propargyl, asrepresented by the compounds herein designated M32 (Appendix IV) and M33(Appendix III), respectively.

In another more preferred embodiment, in the compound of formula II, R₃is a group —N(CH₃)-propargyl, as represented by the compound hereindesignated M30 (Appendix III).

In still another preferred embodiment of the invention, there isprovided a compound of formula III:

wherein

R₉ is selected from the group consisting of (i) H; (ii) —CO—CH₂—R₁;

-   -   (iii) —CH₂—COOCH₂C₆H₅; (iv) —CH(CH₂SH)COOC₂H₅;

R₁₀ is X; —CH₂—CH(SH)COOC₂H₅; or

n is an integer from 1 to 6, preferably 1 or 2;

R₁₂ is X, C₁-C₄ lower alkyl, preferably methyl, COOC₂H₅, or —(CH₂)₂—OH;

and X is a propargyl group.

In one preferred embodiment, R₉ is —CO—CH₂—R₁, wherein R₁ is the residueof an analog of a neuroprotective peptide or a fragment thereofcontaining a Cys residue. In more preferred embodiments, the compoundsof formula III are the hydroxamates containing a propargylamino groupand a R₁ selected from the group consisting of the residue of a VIPfragment analog of SEQ ID NO:2 bearing a stearyl (M6B, Appendix V) or aFmoc group (M7B, Appendix V) at the amino terminal, the residue of aGnRH analog of SEQ ID NO:4 (M8B, Appendix V) or SEQ ID NO:5 (M22B,Appendix V), the residue of a Substance P analog of SEQ ID NO:7 (M27B,Appendix V) or SEQ ID NO:8 (M28B, Appendix V), and the residue of anenkephalin analog of SEQ ID NO:11 (M19B, Appendix V), SEQ ID NO:12(M21B, Appendix V), SEQ ID NO:13 (M18B, Appendix V), and SEQ ID NO:14(M20B, Appendix V).

In other preferred embodiments, R₉, R₁₀ and R₁₂ are as defined above asexemplified by the compounds herein designated M35, M36, M37, M38, M39,M40, M41, M42, M43, M44, M45 and M46 (Appendix V).

In yet another preferred embodiment, there is provided a compound offormula IV:

wherein

R₁₁ is selected from the group consisting of

(i) —S—CH₂—CH(COOH)—NH—X;

(ii) —N(X)—CH₂COO—CH₂—C₆H₅;

(iii) —N(CH₃)—X;

(iv) (iv) —N(X)—CH(CH₂SH)COOC₂H₅;

(v) —CH₂—NH—NH—CO—CH(CH₂OH)—NH—X;

(vi) —C(CH₃)(COOH)—NH—NH—X;

(vii) —CH(COOH)—NH—X;

(viii) —CH(COOC₂H₅)—NH—X; and

R₁₃ is X, —(CH₂)₂—OX, or —COO—(CH₂)₂—NH—X; and

X is a propargyl group.

In preferred embodiments, the pyridinone derivatives are exemplified bythe compounds herein designated M9b, M11b, M12b, M13b, M15b, HLA16b,M17a, HLA20a, M30a, M31a, M33a, and M34b, whose formulas are depicted inAppendix VI herein. Other derivatives of pyridinones may be used whereinthe Me group is replaced by a different alkyl, e.g. ethyl, or the ringmay contain a further alkyl group, optionally substituted.

The following Schemes A-D depict examples of methods that can be usedfor the preparation of compounds of the formulas I, II, III and IV. Allof the starting materials are prepared by procedures described in theseschemes, by procedures well known to one of ordinary skill in organicchemistry or can be obtained commercially. All of the final compounds ofthe present invention are prepared by procedures described in theseschemes or by procedures analogous thereto, which would be well known toone of ordinary skill in organic chemistry. All the modified peptideswere prepared automatically or manually by solid-phase peptide synthesisusing Fmoc chemistry following the company's protocols.

The compounds of formula I can be prepared by the method as shown inScheme A, or by the methods given in the examples or by analogousmethods.

As shown in Scheme A below, 8-hydroxyquinoline (A1) is treated withhydrochloric acid and formaldehyde to give5-chloromethyl-8-hydroxyquinoline (A2). The chloro group of A2 issubstituted with propargylamine to afford5-(1-propargylamino)methyl-8-hydroxyquinoline (A3). Bromination of A3employing N-bromosuccinimide (NBS) provides the bromide A4 which is thentreated, for example, with the modified peptide [D-Cys⁶]GnRH to give thetarget compound A5, which is the compound M22A in Appendix I.

The compounds of formula II can be prepared by the method as shown inScheme B, or by the methods given in the examples or by analogousmethods.

As shown in Scheme B below, substitution of the chloro group in5-chloromethyl-8-hydroxyquinoline A2 (prepared as described in Scheme A)with cysteine provides the intermediate B1. Alkylation of theintermediate B1 with propargyl bromide produces the target compound B2,which is the compound M11a or M12a in Appendix III.

In another synthetic route according to Scheme B, the OH group of5-chloromethyl-8-hydroxyquinoline A2 is first protected as its acetylderivative B3. Condensation of compound B3 with diethylacetamidomalonate proceeds smoothly in ethanol, with sodium ethoxide asthe base, and gives the expected condensation derivative B4.Decarboxylation of compound B4 affords the amino acid derivative B5.Esterification of B5 in the presence of thionyl chloride and ethanolproduces the ethyl ester derivative B6. Compound B6 is hydrolyzed usingα-chymotripsin as a catalyst to give a mixture of the L-amino acidderivative B8 (α-chymotripsin is L-specific and hydrolyzes only theL-form) and the D-amino acid ester derivative B7, which can be separatedby HPLC. Treatment of the L-amino acid B8 or D-amino acid (obtained fromthe ester B7) with propargyl bromide provides the desired compound B9,which is the compound M9a or M31 in Appendix III.

For production of the compounds of formula III, the method shown inScheme C or analogous methods or the methods given in the examples maybe used.

Hydroxamates (C2) can either be purchased from commercial sources orprepared by procedures well known to one of ordinary skill in organicchemistry. Alkylation of a hydroxamate (C2) with propargyl bromide (C1)in the presence of the appropriate base such as diisopropylethylamineprovides a compound C3. Reaction of compound C3 with chloroacetylchloride at about 0° C. in the presence of nitrogen, gives theN-chloroacetyl derivative (C4). Treatment of C4, for example, with themodified peptide [D-Cys⁶]GnRH, followed by deprotection of thehydroxamate, affords the target compound C6, which is the compound M22Bin Appendix V.

The compounds of formula IV can be manufactured by the method as shownin Scheme D, or by the methods given in the examples or by analogousmethods.

The starting material 1-chloroethyl-2-methyl-3-hydroxypyridinone (D1)can be prepared by known procedures. Treatment of D1 with cysteineresults in the formation of the sulfide D2, that is then alkylated withpropargylamine to give the desired compound D4, which is the compounddesignated M11b in Appendix VI.

In another synthetic route according to Scheme D, the compound D1 isdirectly reacted with propargylpiperazine D7 to give the desiredchelator compound D4, which is the compound designated HLA20a inAppendix VI.

Propargylpiperazine (D7) is prepared by reaction of N-Boc-piperazine(D5) with propargylamine giving the compound D6, followed by removal ofthe protecting Boc group using trifluoroacetic acid.

Also contemplated by the present invention are pharmaceuticallyacceptable salts of the compounds of formula I, both salts formed by anycarboxy groups present in the molecule and a base as well as acidaddition and/or base salts.

Pharmaceutically acceptable salts are formed with metals or amines, suchas alkali and alkaline earth metals or organic amines. Examples ofmetals used as cations are sodium, potassium, magnesium, calcium, andthe like. Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamine,N-methylglucamine, and procaine (see, for example, Berge S. M., et al.,“Pharmaceutical Salts,” (1977) J. of Pharmaceutical Science, 66:1-19).The salts can also be pharmaceutically acceptable quaternary salts suchas a quaternary salt of the formula -NRR′R″+Z′ wherein R, R′ and R″ eachis independently hydrogen, alkyl or benzyl and Z is a counterion,including chloride, bromide, iodide, O-alkyl, toluenesulfonate,methylsulfonate, sulfonate, phosphate, or carboxylate.

Pharmaceutically acceptable acid addition salts of the compounds includesalts derived from inorganic acids such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorous, and the like,as well as salts derived from organic acids such as aliphatic mono- anddicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoicacids, alkanedioic acids, aromatic acids, aliphatic and aromaticsulfonic acids, etc. Such salts thus include sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide, acetate, propionate,caprylate, isobutyrate, oxalate, malonate, succinate, suberate,sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate,methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate,toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate,methanesulfonate, and the like. Also contemplated are salts of aminoacids such as arginate and the like and gluconate or galacturonate (see,for example, Berge S. M., et al., “Pharmaceutical Salts,” (1977) J. ofPharmaceutical Science, 66:1-19).

The acid addition salts of said basic compounds are prepared bycontacting the free base form with a sufficient amount of the desiredacid to produce the salt in the conventional manner. The free base formmay be regenerated by contacting the salt form with a base and isolatingthe free base in the conventional manner. The free base forms differfrom their respective salt forms somewhat in certain physical propertiessuch as solubility in polar solvents, but otherwise the salts areequivalent to their respective free base for purposes of the presentinvention.

The base addition salts of said acidic compounds are prepared bycontacting the free acid form with a sufficient amount of the desiredbase to produce the salt in the conventional manner. The free acid formmay be regenerated by contacting the salt form with an acid andisolating the free acid in the conventional manner. The free acid formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but otherwise the saltsare equivalent to their respective free acid for purposes of the presentinvention.

The compounds of the invention are specific iron chelators that aresuitable to bind unbound iron within the cells. Iron that is not boundto transferrin is the toxic form of iron. The iron chelators of theinvention have good transport properties and cross cell membranes thuschelating the unbound iron in excess within the cells. It is expectedthat their complexes with iron will leave the cells freely and will berapidly excreted. It is further expected that the compounds, or at leasta major part of the compounds, will be able to cross the BBB and thuswill be suitable candidates for treatment of neurodegenerative diseases,disorders and conditions.

In another aspect, the present invention relates to pharmaceuticalcompositions comprising a compound of the invention or apharmaceutically acceptable salt thereof, in combination with apharmaceutically acceptable carrier.

In a further aspect, the present invention provides the use of acompound of the invention or of a pharmaceutically acceptable saltthereof as neuroprotective iron chelator for the preparation of apharmaceutical composition for iron chelation therapy.

The pharmaceutical composition are intended for use in iron chelationtherapy for treatment of diseases, disorders and conditions associatedwith iron overload and oxidative stress.

The pharmaceutical composition of the invention is for treatment and/orprevention of diseases, disorders and conditions associated with ironoverload and oxidative stress such as, but not limited to,neurodegenerative and cerebrovascular diseases and disorders, neoplasticdiseases, hemochromatosis, thalassemia, cardiovascular diseases,diabetes, inflammatory disorders, anthracycline cardiotoxicity, viral,protozoal and yeast infections, and for retarding ageing, and preventionand/or treatment of skin ageing and skin protection against sunlightand/or UV light.

In one preferred embodiment, the pharmaceutical compositions are for usefor iron chelation and neuroprotection in the prevention and/ortreatment of neurodegenerative and cerebrovascular diseases, conditionsand disorders such as Parkinson's disease, Alzheimer's disease, stroke,amyotrophic lateral sclerosis (ALS), multiple sclerosis, Friedreich'sataxia, Hallervorden-Spatz disease, epilepsy and neurotrauma. In onepreferred embodiment, the pharmaceutical composition is for treatment ofParkinson's disease. In another preferred embodiment, the pharmaceuticalcomposition is for treatment of Alzheimer's disease. In a furtherpreferred embodiment, the pharmaceutical composition is for treatment ofa cerebrovascular disorder, particularly stroke.

The “prevention’ aspect of the use of the iron chelators of theinvention in diseases such as Parkinson's disease and Alzheimer'sdisease involves the prevention of further neurodegeneration and thefurther progress of the disease.

In still another preferred embodiment, the pharmaceutical compositionsare for inhibition of cell proliferation in the treatment of neoplasticdiseases, all types of cancer being encompassed by the invention. Theiron chelator of the invention can be used alone or in combination withone or more cytotoxic anticancer drugs.

In another preferred embodiment, the pharmaceutical compositions are forprevention and/or treatment of iron overload in hemochromatosis andthalassemia.

In yet another preferred embodiment, the pharmaceutical compositions arefor prevention and/or treatment of cardiovascular diseases, e.g. toprevent the damage associated with free radical generation inreperfusion injury.

In yet another preferred embodiment, the pharmaceutical compositions arefor prevention and/or treatment of diabetes.

In yet another preferred embodiment, the pharmaceutical compositions arefor prevention and/or treatment of inflammatory disorders. In onepreferred embodiment, the inflammatory disorder is a joint inflammatorydisorder, particularly rheumatoid arthritis. In another preferredembodiment, the inflammatory disorder is inflammatory bowel disease(IBD). In a further preferred embodiment, the inflammatory disorder ispsoriasis.

In yet another preferred embodiment, the pharmaceutical compositions arefor prevention and/or treatment of anthracycline cardiotoxicity, in caseof cancer patients being treated with anthracycline neoplastic drugs.

In yet another preferred embodiment, the pharmaceutical compositions arefor prevention and/or treatment of viral, protozoal and yeastinfections. In one preferred embodiment, the viral infection is aretroviral infection, e.g. HIV-1, and the compound is used in thetreatment of AIDS, optionally in combination with antiviral agents. Inanother preferred embodiment, the protozoal infection is malaria causedby Plasmodium falciparum. In a further preferred embodiment, the yeastinfection is a Candida albicans infection.

In yet another preferred embodiment, the pharmaceutical compositions arefor retarding ageing and/or improving the ageing process by preventionof ageing-related diseases, disorders or conditions such asneurodegenerative diseases, disorders or conditions.

In yet another preferred embodiment, the pharmaceutical compositions arefor prevention and/or treatment of skin ageing and/or skin damageassociated with ageing and/or exposure to sunlight and/or UV light.

In yet another preferred embodiment, the present invention provides acosmetic composition for topical application for prevention and/ortreatment of skin ageing and/or skin damage associated with ageingand/or exposure to sunlight and/or UV light. The cosmetic compositionmay be in the form of a lotion or cream and may be administered withother agents for skin treatment.

In another embodiment, the iron chelators are for use ex-vivo forpreservation of organs intended for transplantation such as heart, lungor kidney

In still another aspect, the present invention provides a method foriron chelation therapy which comprises administering to an individual inneed thereof an effective amount of a compound of the invention or of apharmaceutically acceptable salt thereof.

In one embodiment, the present invention provides a method for theprevention and/treatment of a neurodegenerative disease, condition ordisorder, which comprises administering to an individual in need thereofan effective amount of a compound of the invention or of apharmaceutically acceptable salt thereof.

In yet another embodiment, the present invention provides a method forthe prevention and/or treatment of cancer, which comprises administeringto an individual in need thereof an effective amount of a compound ofthe invention or of a pharmaceutically acceptable salt thereof. In onepreferred embodiment, the iron chelator of the invention is administeredbefore, concurrently or after administration of one or morechemotherapeutic agents.

In another embodiment, the present invention provides a method for theprevention and/or treatment of iron overload in hemochromatosis orthalassemia patients, which comprises administering to said patient aneffective amount of a compound of the invention or of a pharmaceuticallyacceptable salt thereof.

In yet another preferred embodiment, the present invention provides amethod for prevention and/or treatment of cardiovascular diseases, e.g.to prevent the damage associated with free radical generation inreperfusion injury, which comprises administering to an individual inneed thereof an effective amount of a compound of the invention or of apharmaceutically acceptable salt thereof.

In yet another preferred embodiment, the present invention provides amethod for prevention and/or treatment of diabetes, which comprisesadministering to an individual in need thereof an effective amount of acompound of the invention or of a pharmaceutically acceptable saltthereof.

In yet another preferred embodiment, the present invention provides amethod for prevention and/or treatment of inflammatory disorders, whichcomprises administering to an individual in need thereof an effectiveamount of a compound of the invention or of a pharmaceuticallyacceptable salt thereof. In one preferred embodiment, the inflammatorydisorder is a joint inflammatory disorder, particularly rheumatoidarthritis. In another preferred embodiment, the inflammatory disorder isinflammatory bowel disease (IBD). In a further preferred embodiment, theinflammatory disorder is psoriasis.

In yet another preferred embodiment, the present invention provides amethod for prevention and/or treatment of anthracycline cardiotoxicity,which comprises administering to an individual undergoing treatment withanthracycline neoplastic drugs an effective amount of a compound of theinvention or of a pharmaceutically acceptable salt thereof.

In yet another preferred embodiment, the present invention provides amethod for prevention and/or treatment of a viral, protozoal or yeastinfection which comprises administering to an individual in need thereofan effective amount of a compound of the invention or of apharmaceutically acceptable salt thereof. In one preferred embodiment,the viral infection is a retroviral infection, e.g. HIV-1, and thecompound is used in the treatment of AIDS, optionally in combinationwith antiviral agents. In another preferred embodiment, the protozoalinfection is malaria caused by Plasmodium falciparum. In a furtherpreferred embodiment, the yeast infection is a Candida albicansinfection.

In yet another preferred embodiment, the present invention provides amethod for retarding ageing and/or improving the ageing process byprevention of ageing-related diseases, disorders or conditions whichcomprises administering to an individual in need thereof an effectiveamount of a compound of the invention or of a pharmaceuticallyacceptable salt thereof. The individual in need may be a healthyindividual or an individual suffering from an age-related disease suchas a neurodegenerative disease, disorder or condition.

In yet another preferred embodiment, the present invention provides amethod for prevention and/or treatment of skin ageing and/or skin damageassociated with ageing and/or exposure to sunlight and/or UV light,which comprises administering to an individual in need thereof aneffective amount of a compound of the invention or of a pharmaceuticallyacceptable salt thereof. The compound is most preferably administeredtopically in a pharmaceutical or cosmetic formulation.

The present invention also provides the use of the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline (hereinidentified as VK-28, Appendix IV) for the preparation of apharmaceutical composition for treatment and/or prevention of a disease,disorder or condition associated with iron overload and oxidative stressselected from a neoplastic disease, hemochromatosis, thalassemia, acardiovascular disease, diabetes, a inflammatory disorder, anthracyclinecardiotoxicity, a viral infection, a protozoal infection, a yeastinfection, retarding ageing, and prevention and/or treatment of skinageing and skin protection against sunlight and/or UV light.

The present invention further provides a method for iron chelationtherapy which comprises administering to an individual in need thereofan effective amount the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline (hereinidentified as VK-28, Appendix IV) for treatment and/or prevention of adisease, disorder or condition associated with iron overload andoxidative stress, excluding the prevention and/or treatment of aneurodegenerative disease, condition or disorder.

The present invention still further provides the use of the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline (hereinidentified as VK-28, Appendix IV) for the preparation of a cosmeticcomposition for topical application for prevention and/or treatment ofskin ageing and/or skin damage associated with ageing and/or exposure tosunlight and/or UV light.

The present invention yet further provides the use of the compound5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline (hereinidentified as VK-28, Appendix IV) ex-vivo for preservation of organsintended for transplantation such as heart, lung or kidney.

For preparing the pharmaceutical compositions of the present invention,methods well-known in the art can be used. Inert pharmaceuticallyacceptable carriers can be used that are either solid of liquid. Solidform preparations include powders, tablets, dispersible granules,capsules, cachets and suppositories.

A solid carrier can be one or more substances which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or tablet disintegrating agents; it can also be anencapsulating material.

Liquid pharmaceutical compositions include solutions, suspensions, andemulsions. As an example, water or water-propylene glycol solutions forparenteral injection may be mentioned. Liquid preparations can also beformulated in solution in aqueous polyethylene glycol solution. Aqueoussolutions for oral use can be prepared by dissolving the activecomponent in water and adding suitable colorants, flavoring agents,stabilizers, and thickening agents as desired. Aqueous suspensions fororal use can be made by dispersing the finely divided active componentin water with viscous material, i.e., natural or synthetic gums, resins,methyl cellulose, sodium carboxymethyl cellulose, and other well-knownsuspending agents.

Preferably, the pharmaceutical composition is in unit dosage form. Insuch form, the preparation is subdivided into unit doses containingappropriate quantities of the active component. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation, for example, packeted tablets, capsules, and powders invial or ampoules. The unit dosage form can also be a capsule, cachet, ortable itself or it can be the appropriate number of any of thesepackaged forms.

In therapeutic use for the treatment of Parkinson's disease, thecompounds utilized in the pharmaceutical method of this invention may beadministered to the patient at dosage levels of from 1 mg/Kg to 20 mg/Kgper day.

In therapeutic use for the treatment of stroke one or more dosages offrom about 100 mg/Kg to about 500 mg/Kg of body weight may beadministered to the patient as soon as possible after the event.

The dosage, however, may be varied depending upon the requirements ofthe patient, the severity of the condition being treated, and thecompound being employed. Determination of optimum dosages for aparticular situation is within the skill of the art.

The following examples illustrate particular methods for preparingcompounds in accordance with this invention. These examples are intendedas an illustration, and not as a limitation, of the scope of theinvention.

EXAMPLES

The following examples describe the synthesis of the compounds of theinvention (Chemical Section) and their biological activity (BiologicalSection).

I. Chemical Section

(i) Appendices I-VII

The structural formulas of the compounds of the invention are depictedin Appendices I to VI herein. Appendix VII contains the formulas of someintermediate compounds. For better identification of the compounds inthe following examples, when possible, the compound designations usedherein and the respective Appendix are given within brackets near thecompound's name. When suitable, the designation given in the Schemes A-Dabove are used for starting compounds or intermediates.

The contents of the Appendices are as follows:

Appendix I—Compounds M6A, M7A, MBA, M18A, M19A, M20A, M21A, M22A, M27A,M28A. Appendix II—Compounds M6, M7, M8, M11, M12, M18, M19, M20, M21,M22 M27, M28. Appendix III—Compounds HLA16a, HLA20, M9a, M10a, M11a,M12a, M13a, M15a, M17, M30, M31, M33, M34.

Appendix IV—Compounds VK-28 (known), HLA16, HLM7, HLM8, HLM9, M9, M10,M11B, M12B, M13, M15, M32.

Appendix V—Compounds M6B, M7B, M8B, M18B, M19B, M20B, M21B, M22B, M27B,M28B, M35, M36, M36a, M37-46. Appendix VI—Compounds M9b, M11b, M12b,M13b, M15b, HLA16b, M17a, HLA20a, M30a, M31a, M33a, M34b. AppendixVII—Intermediates H1, H2, H3, H4, H5, H6. (ii) General

Starting materials for chemical synthesis were obtained from thefollowing companies: Aldrich (USA), E. Merck (Germany), Fluka,(Switzerland).

Proton NMR spectra were measured on a Bruker WH-270, a Bruker DPX-250,or a Bruker AMX-400 NMR spectrometer. Flash column chromatographyseparations were performed on silica gel Merck 60 (230-400 mesh ASTM).UV/VIS spectra were measured on a Hewlett-Packard 8450A diode arrayspectrophotometer. TLC was performed on E. Merck Kieselgel 60 F₂₅₄plates. Staining of TLC plates was done by: (i) basic aqueous 1% KMnO₄;(ii) 0.3% ninhydrin in EtOH_(abs). Tetrahydrofuran was distilled overLiAlH₄ and passed through an Al₂O₃ column. Mass spectra (DI, EI-MS) weremeasured on a VG-platform-II electrospry single quadropole massspectrometry (Micro Mass, UK).

EXAMPLES Example 1 Synthesis ofD-N-propargyl-3-(8-hydroxyquinolin-5-yl)alanine (M9a, Appendix III) andL-N-propargyl-3-(8-hydroxyquinolin-5-yl)alanine (M10a, Appendix III)

These enantiomers of Formula II herein were prepared by the methoddepicted in Scheme B above, by the following steps:

(i) Synthesis of 5-chloromethyl-8-hydroxyquinolinoline (A2)

A mixture of 14.6 g (0.1 mole) of 8-hydroxyquinolinoline, 16 ml of 32%hydrochloric acid, and 16 ml (0.1 ml) of 37% formaldehyde at 0° C. wastreated with hydrogen chloride gas for 6 h. The solution was allowed tostand at room temperature for 2 h without stirring. The yellow solid wascollected on a filter and dried to give crude5-chloromethyl-8-hydroxyquinoline hydrochloride (A2): 2 16 g; H¹ NMR(250 MHz, CDCl₃): 5.32 (s, 2H), 7.53 (m, 1H), 7.85 (m, H), 8.12 (m, 1H),9.12 (m, 1H), 9.28 (m, 1H).

(ii) Synthesis of 8-(5-chloromethyl)quinolyl acetate (B3)

To a stirred solution of crude A2 obtained in step (i) above (230 mg, 1mmole) in dry DMF (5 ml) at 0° C., pyridine (0.3 ml, 2.5 mmol) andacetyl chloride (0.6 ml, 8 mmole) were slowly added simultaneously underN₂. The reaction mixture was stirred at 0° C. for 1 h and at roomtemperature for 1 h. After cooling to 0° C., 10 ml of water were added,and the resulting mixture was stirred at 0° C., for 20 min. The mixturewas then extracted with chloroform (3×20 ml). The combined organic layerwas washed with saturated NaHCO₃ (2×20 ml), brine (2×20 ml) and driedover Na₂SO₄. Evaporation afforded the compound (B3) (crude) (180 mg,75%) as a light brown solid. m.p.=119-120° C., R_(f) 0.75 (CHCl₃: MeOH:NH₃ 9: 1: 0.25). ¹H NMR (250 MHz, CDCl₃): 2.25 (s, 3H), 4.98 (s, 2H),7.40 (d, J=7.7 Hz, 1H), 7.55 (m, 2H), 8.47 (dd, J=8.6, 1.6 Hz, 1H), 8.96(dd, J=4.2, 1.5 Hz, 1H).

(iii) Synthesis of diethyl8-hydroxyquinolin-5-yl-methyl-acetamidomalonate (HLM7, Appendix IV)

To a solution of acetamidomalonate (183 mg, 0.842 mmol) and metallicsodium (34 mg, 0.842 mmole) in ethanol (50 ml),8-(5-chloromethyl)quinolyl acetate (B3) (180 mg, 765 mmole) in CHCl₃ (5m) was added. The mixture was stirred for 5 h under reflux, cooled, andevaporated in vacuum. Water (20 ml) was added to the residue and themixture was extracted with CHCl₃: EtOAc (3×20 ml, 1: 1). The organiclayer was washed with brine (2×20 ml) and dried over Na₂SO₄. Evaporationafforded the title compound (HLM7): (170 mg, 59%) as a light brown solid(crude) m.p.=153-154° C., R_(f) 0.25 (CHCl₃: MeOH: NH₃ 9: 1: 0.25). ¹HNMR (250 MHz, CDCl₃): 1.30 (dd, J=7.1, 7.1 Hz, 6H), 1.86 (s, 3H), 4.03(s, 2H), 4.27 (m, 4H), 6.46 (s, 1NH), 7.10 (dd, J=16.6, 7.9 Hz, 2H),7.42 (dd, J=8.7, 4.2 Hz, 1H), 8.76 (dd, J=4.2, 1.1 Hz, 1H).

(iv) Synthesis of DL-3-(8-hydroxyquinolin-5-yl)alanine (HLM8, AppendixIV)

Diethyl (8-hydroxyquinolin-5-yl-methyl)-acetamidomalonate (HLM7)obtained in (iii) above (8.9 g, 21.9 mmol) was dissolved in 6 N HCl (150ml), and the resultant mixture was refluxed for 10 h. The reactionmixture was evaporated to dryness; solvent was removed in vacuum. Theresidue was redissolved in H₂O and filtered. The pH of the solution wasadjusted to between 5-5.5 with 10% NaOH. A yellow precipitate wascollected by filtration, washed with water thoroughly, andre-crystallized from water (pH=5.5) and then washed with acetone to givethe title compound (HLM8) (3.8 g, yield 65%), 100% purity, checked byHPLC [(C₁₈; solvent A=water, 0.1% v/v TFA; solvent B=MeCN: water=3: 1,0.1% v/v TFA; t_(R)=18.2 min (linear gradient 0-80% B over 55 min)].m.p.=194° C., (decompose). ¹H NMR (250 MHz, D₂O) 3.42 (m, 1H), 3.58 (m,1H), 3.93 (dd, J=7.2, 7.2 Hz, 1H), 7.22 (d, J=8.0 Hz, 1H), 7.50 (d,J=8.1 Hz, 1H), 7.94 (dd, J=8.7, 5.4 Hz, 1H), 8.84 (d, J=5.1 Hz, 1H),9.06 (d, J=8.7 Hz, 1H); ¹³C NMR (100 MHz, DMSO) 32.14, 53.11, 111.03,121.27, 127.46, 129.76, 133.35, 138.13, 147.46, 152.55, 170.30. Massspectrometry: calculated for C₁₂H₁₂N₂O₃ m/z [M+H]⁺=233.24. found[M+H]⁺=233.25.

(v) Synthesis of DL-3-(8-hydroxyquinolin-5-yl)alanine ethyl ester (HLM9,Appendix IV)

To a stirred slurry of DL-3-(8-hydroxyquinolin-5-yl)alanine (HLM8) (2.19g, 7.04 mmol) in absolute ethanol (26 ml) at 0° C., with protection fromatmospheric moisture by CaCl₂ tube in N₂, thionyl chloride was addeddropwise (1.1 ml, 14.1 mmol). The reaction mixture was stirred at 0° C.for 30 minutes and at room temperature for 30 minutes, and then refluxedovernight. The solution was evaporated to dryness in vacuum. The residuewas dissolved in absolute ethanol and reevaporated to dryness. To ensurecompleteness of the esterification, the whole operation was repeated.The ester HLM9 (yellow solid, 1.67 g, 92% yield), which was shown byHPLC to contain about 1% of free amino acid, was further purified byFC(CH₂Cl₂: MeOH: AcOH 9: 1.5: 1.5). ¹H NMR (250 MHz, D₂O) 0.94 (dd,J=7.2, 7.2 Hz, 3H), 3.63 (m, 2H), 4.01 (m, 2H), 4.34 (dd, J=7.7, 7.5 Hz,1H), 7.31 (d, J=8.0 Hz, 1H), 7.56 (d, J=8.2 Hz, 1H), 8.01 (dd, J=8.7,5.4 Hz, 1H), 8.93 (d, J=5.4 Hz, 1H), 9.08 (d, J=8.7 Hz, 1H).

(vi) Synthesis of L-3-(8-hydroxyquinolin-5-yl)alanine (M10, Appendix 19and D-3-(8-hydroxy-quinolin-5-yl)alanine ethyl ester (B7, Scheme B)

DL-3-(8-hydroxy-quinolin-5-yl)alanine ethyl ester (HLM9) (89.2 mg)obtained in 1(v) above was dissolved in 5 ml water, the pH of thesolution was adjusted to about 6.4 with 0.2M NaOH, α-chymotrypsin (0.9mg) was added, and the mixture was incubated at room temperature for 6h, the pH being kept constant by addition of 0.2 M NaOH. After thedigestion, the mixture was lyophilized to dryness and separated bysemi-preparative HPLC [(C₁₈; solvent A=water, 0.1% v/v TFA; solventB=MeCN: water=3: 1, 0.1% v/v TFA; t_(R)=18.0 min (linear gradient 0-80%B over 55 min)]. Since α-chymotrypsin is specific for the L-form,L-3-(8-hydroxyquinolin-5-yl)alanine (M10) was obtained: 27.6 mg, 35%,100% purity, [α]_(D) ²⁰=+13.5. (D-3-(8-hydroxy-quinolin-5-yl)alanineethyl ester (B7) was recovered: 39.2 mg, 88% recovery). ¹H NMR (250 MHz,D₂O) 3.49 (m, 1H), 3.65 (m, 1H), 3.95 (m, 1H), 7.34 (m, 1H), 7.57 (m,1H), 8.0 (m, 1H), 8.91 (m, 1H), 9.14 (m, 1H). Mass spectrometry:calculated for C₁₂H₁₂N₂O₃ m/z [M+H]⁺=233.24. found [M+H]⁺=233.26.

(vii) Synthesis of D-3-(8-hydroxyquinolin-5-yl)alanine (M9, Appendix IV)

For hydrolysis of the ethyl ester, 33.4 mgD-3-(8-hydroxyquinolin-5-yl)alanine ethyl ester (B7) recovered in step1(vi) was dissolved in 11 ml 0.2 M NaOH, and the solution was stirred atroom temperature for 6 h The water was removed by lyophilization, andthe product was purified by semi-preparative HPLC [(C₁₈; solventA=water, 0.1% v/v TFA; solvent B=MeCN: water=3: 1, 0.1% v/v TFA;t_(R)=18.4 min (linear gradient 0-80% B over 55 min)] to give the titlecompound M9: 26.8 mg, 89%, 99.1% purity. [α]_(D) ²⁰=−10.3. ¹H NMR (250MHz, D₂O) 3.40 (s, 1H), 3.55 (s, 1H), 3.91 (s, 1H), 7.15 (s, 1H), 7.45(s, 1H), 7.91 (s, 1H), 8.80 (s, 1H), 9.02 (s, 1H). Mass spectrometry çcalculated for C₁₂H₁₂N₂O₃ m/z [M+H]⁺=233.24. found [M+H]⁺=233.26.

(viii) Synthesis of D-N-propargyl-3-(8-hydroxyquinolin-5-yl)alanine(M9a, Appendix III)

A mixture of NaHCO₃ (17 mg, 0.2 mmol) and compound M9 (23.2 mg, 0.1mmol) from step (vii) above was dissolved in 5 ml DMSO, and the solutionwas stirred at room temperature for 2 h. To the solution, propargylbromide (11.9 mg, 0.1 mmol) was slowly added, and the solution wasstirred at room temperature for 24 h. The solvent was removed by vacuum,and the crude product was purified by semi-preparative HPLC [(C₁₈;solvent A=water, 0.1% v/v TFA; solvent B=MeCN: water=3: 1, 0.1% v/v TFA;t_(R)=22.4 min (linear gradient 0-80% B over 55 min)] to give the titlecompound M9a: 19 mg, 70% yield. [α]_(D) ²⁰=−12.3. H¹ NMR (250 MHz,CDCl₃), 2.21 (m, 1H), 3.28 (d, J=2.44 Hz, 2H), 3.82 (s, 2H), 7.08 (m,1H), 7.33 (d, J=10.59, 1H), 7.46 (dd, J=8.55, 4.18 Hz, 1H), 8.67 (dd,J=8.56, 1.55 Hz, 1H), 8.78 (dd, J=4.18. 1.51 Hz, 1H). Mass spectrometry:calculated for C₁₅H₁₄N₂O₃ m/z [M+H]⁺=271.28. found [M+H]⁺=271.30.

(ix) Synthesis of L-N-propargyl-3-(8-hydroxyquinolin-5-yl)alanine (M10a,Appendix III)

The title compound was synthesized using the procedure of step (viii)above. 78% yield. [α]_(D) ²⁰=+15.3. H¹ NMR (250 MHz, CDCl₃), 2.25 (m,1H), 3.26 (d, J=2.44 Hz, 2H), 3.80 (s, 2H), 7.08 (m, 1H), 7.33 (d,J=10.59, 1H), 7.46 (dd, J=8.55, 4.18 Hz, 1H), 8.64 (dd, J=8.53, 1.55 Hz,1H), 8.78 (dd, J=4.18. 1.51 Hz, 1H); Mass spectrometry: calculated forC₁₅H₁₄N₂O₃ m/z [M+H]⁺=271.28. found [M+H]⁺=271.30.

Example 2 Synthesis of5-(4-propargylpiperazin-1-ylmethyl)-8-hydroxy-quinoline (HLA20, AppendixIII)

The title compound was prepared by reaction of5-chloromethyl-8-hydroxyquinolinoline (A2) with N-propargyl piperazineas follows:

(i) Synthesis of tert-butyl 1-piperazinecarboxylate (D5, Scheme D)

A solution of di(tert-butyl) dicarbonate (2.93 g, 12.77 mmole) in MeOH(25 ml) was slowly added to a stirring solution of piperazine (2.00 g,23.22 mmole) in MeOH (50 ml) at 0° C. The mixture was then stirred for 2days at room temperature, and the solvent was removed in vacuum. Thecrude solid was redissolved in Et₂O (100 ml) with warming, and a whiteprecipitate was filtered off. The product was extracted from the motherliquor with 1 M citric acid solution (3×50 ml), and the aqueous layerwas washed with Et₂OAc (3×50 ml), basified with Na₂CO₃ (pH 11), andextracted with Et₂OAc (3×50 ml). The organic layer was dried over Na₂SO₄and evaporated in vacuum to give tert-butyl 1-piperazinecarboxylate (D5)as a waxy white solid (crude, 1.57 g, 66%), mp=53-45° C. H¹ NMR (250MHz, CDCl₃) 1.42 (s, 9H), 1.89 (s, 1NH), 2.78 (m, 4H), 3.36 (m, 4H).

(ii) Synthesis of tert-butyl 4-propargylpiperazine-1-carboxylate (D6,Scheme D)

Propargyl bromide (356.9 mg, 3 mmol) was slowly added to a mixture oftert-butyl 1-piperazinecarboxylate obtained in step 2(i) above (558.8mg, 3 mmole) and diisopropylethylamine (407.1 mg, 3.15 mmol) in CHCl₃(25 ml) at 0° C. The mixture was stirred for 24 h at room temperature.50 ml of CHCl₃ was then added and the solution was washed with 5% NaHCO₃(3×50 ml), brine (2×50 ml), and then dried over Na₂SO₄. The solution wasfiltered and evaporated to dryness. The residue was crystallized from amixture of benzene-hexane (1:1) to give tert-butyl4-propargylpiperazine-1-carboxylate (D6) (crude 337 mg, 86%). H¹ NMR(250 MHz, CDCl₃): 1.42 (s, 9H), 2.22 (s, 1H), 2.46 (s, 4H), 3.26 (s,2H), 3.41 (s, 4H).

(iii) Synthesis of N-propargylpiperazine (D7, Scheme D)

tert-Butyl 4-propargylpiperazine-1-carboxylate of step 2(ii) above (570mg, 2.545 mmol) was dissolved in trifluoroacetic acid (10 ml) and water(2.5 ml). The mixture was then stirred at room temperature overnight.The solution was evaporated to dryness in vacuum. The residue wasdissolved in water (10 ml) and then basified with Na₂CO₃ (pH 11), andextracted with Et₂OAc (3×50 ml). The organic layer was washed with brine(2×50 ml) and dried over Na₂SO₄ overnight. Evaporation in vacuum gaveN-propargylpiperazine (D7) as white solid. (crude, 193 mg, 62% yield).H¹ NMR (250 MHz, CDCl₃) 1.64 (s, 1NH), 2.26 (m, 1H), 2.55 (dd, J=4.73,4.50 Hz, 4H), 2.93 (dd, J=4.96, 4.84 Hz, 4H), 3.29 (d, J=2.44 Hz, 2H).

(iv) Synthesis of 5-(4-propargylpiperazin-1-ylmethyl)-8-hydroxyquinoline(HLA20, Appendix III))

To a mixture of 5-chloromethyl-8-hydroxyquinoline hydrochloride (A2)(323 mg, 1.407 mmol) and diisopropylethylamine (0.26 ml, 1.477 mmol,1.05 eq) in 6 ml CHCl₃ at 0° C., N-propargylpiperazine obtained in step2(iii) above was added (173 mg, 1.407 mmol, 1 eq). The mixture wasstirred for 24 h at room temperature. 10 ml of CHCl₃ was then added andthe solution was washed with 5% NaHCO₃ (3×50 ml), brine (2×50 ml), andthen dried over Na₂SO₄. The crude product was purified by FC to give thetitle compound (HLA20) as a white solid. (337 mg, 86% yield). H¹ NMR(250 MHz, CDCl₃), 2.23 (m, 1H), 2.54 (s, 8H), 3.28 (d, J=2.44 Hz, 2H),3.80 (s, 2H), 7.08 (m, 1H), 7.36 (d, J=10.59, 1H), 7.46 (dd, J=8.55,4.18 Hz, 1H), 8.67 (dd, J=8.56, 1.55 Hz, 1H), 8.78 (dd, J=4.18. 1.51 Hz,1H); ¹³C NMR (100 MHz, CDCl₃) 46.77, 51.98, 52.87, 60.55, 73.05, 78.92,108.60, 121.40, 124.58, 127.87, 128.87, 134.10, 138.68, 147.50, 151.78.

Example 3 Synthesis of5-(4-propargylaminoethoxycarbonyl-piperazin-1-ylmethyl)-8-hydroxyquinoline(HLA16a, Appendix III) (i) Synthesis of ethyl4-(8-hydroxyquinolin-5-ylmethyl)-1-piperazine carboxylate (HLA16,Appendix IV)

To a mixture of 5-chloromethyl-8-hydroxyquinoline hydrochloride (A2)(2.36 g, 10.2 mmol) and diisopropylethylamine (3.6 ml, 20.4 mmol, 2 eq)in 50 ml CHCl₃ at 0° C., ethyl 1-piperazinecarboxylate (1.5 ml, 10.2mmol, 1 eq) was added. The mixture was stirred for 24 h at roomtemperature, and then 100 ml of CHCl₃ was added and the solution waswashed with 5% NaHCO₃ (3×50 ml), brine (2×50 ml), and then dried overNa₂SO₄. The solution was filtered and evaporated to dryness. The residuewas crystallized from a mixture of benzene-hexane (1:1) to yield thetitle compound HLA16 as white solid. (1.38 g, 42% yield, m.p.=92-93°C.). H¹ NMR (250 MHz, CDCl₃), 1.25 (dd, J=7.1, 7.1 Hz 3H), 2.42 (s, 4H),3.43 (s, 4H), 3.81 (s, 2H), 4.14 (dd, j=14.21, 7.12 Hz, 2H), 7.08 (d,J=7.72 Hz, 1H), 7.31 (m, 1H), 7.47 (dd, J=8.52, 4.20 Hz, 1H), 8.66 (dd,J=8.56, 1.58 Hz, 1H), 8.79 (dd, J=4.18. 1.54 Hz, 1H).

(ii) In an alternative method, the title compound HLA16a is prepared byreaction of 5-chloromethyl-8-hydroxyquinoline withN-propargylaminoethoxycarbonyl-piperazine as follows:

(iia) Synthesis of N-propargylaminoethoxycarbonylpiperazine (H1,Appendix VII)

2-chloroethyl chloroformate (429 mg, 3 mmol) was added slowly to amixture of tert-butyl 1-piperazinecarboxylate obtained as step (ii) inExample 2 (559 mg, 3 mmol) and diisopropylethylamine (407 mg, 3.15 mmol)in CHCl₃ (25 ml) at 0° C. The mixture was stirred for 2 h at roomtemperature. CHCl₃ (50 ml) was then added and the solution was washedwith 5% NaHCO₃ (3×50 ml), brine (2×50 ml), and then dried over Na₂SO₄.The solution was filtered and evaporated to dryness. The residue wasdissolved in CH₂Cl₂ (25 ml) at room temperature, and propargylamine(0.205 ml, 165 mg, 3 mmol) was added. After 24 h of stirring at roomtemperature, the solvent was removed by vacuum, and to the residue wasadded 20 ml of a solution of TFA:H₂O:TES:thioanisole (85:5:5:5). Themixture was then stirred at room temperature for 2 h. The solution wasevaporated to dryness in vacuum. The resulted residue was dissolved inwater (10 ml) and then basified with Na₂CO₃ (pH 11), and extracted withEt₂OAc (3×50 ml). The organic layer was washed with brine (2×50 ml) anddried over Na₂SO₄ overnight. Evaporation in vacuum gave the titlecompound H1: (251 mg, 40% total yield). H¹ NMR (250 MHz, CDCl3+D₂O) 2.26(m, 1H), 3.11 (s 2H), 3.29 (dd, J=10.21, 7.45 Hz, 2H), 4.29 (d, J=11.23,7.12 Hz, 2H), 2.55 (dd, J=4.73, 4.50 Hz, 4H), 2.93 (dd, J=4.96, 4.84 Hz,4H). Mass spectrometry: calculated for C₁₀H_(n)N₃O₂ m/z [M+H]⁺=212.26.found [M+H]⁺=212.22.

(iib) Synthesis of5-(4-propargylaminoethoxycarbonyl-piperazin-1-ylmethyl)-8-hydroxyquinoline(HLA16a, Appendix III)

To a mixture of 5-chloromethyl-8-hydroxyquinoline hydrochloride (A2)(194 mg, 1 mmol) and diisopropylethylamine (0.348 ml, 2 mmol, 2 eq) in 5ml CHCl₃ at room temperature, was added H1 (221 mg, 1 mmol, 1 eq). Themixture was stirred for 24 h at room temperature. 10 ml of CHCl₃ wasthen added and the solution was washed with 5% NaHCO₃ (3×10 ml), brine(2×10 ml), and then dried over Na₂SO₄. The solution was filtered andevaporated to dryness. The residue was crystallized from a mixture oftoluene-hexane (1:1) to yield the title compound HLA16a as white solid.(184 mg, 50% yield). H¹ NMR (250 MHz, CDCl₃+D₂O) 2.23 (m, 1H), 3.30 (s2H), 3.29 (dd, J=10.21, 7.45 Hz, 2H), 4.29 (d, J=11.23, 7.12 Hz, 2H),2.55 (dd, J=4.73, 4.50 Hz, 4H), 2.93 (dd, J=4.96, 4.84 Hz, 4H), 3.78 (s,2H), 7.08 (d, J=7.72 Hz, 1H), 7.31 (m, 1H), 7.47 (dd, J=8.52, 4.20 Hz,1H), 8.66 (dd, J=8.56, 1.58 Hz, 1H), 8.79 (dd, J=4.18, 1.54 Hz, 1H).Mass spectrometry: calculated for C₂₀H₂₄N₄O₃ m/z [M+H]⁺=369.43. found[M+H]⁺=369.28.

Example 4 Synthesis of N-propargylS-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine (M12a, Appendix III) andN-propargyl S-(8-hydroxyquinolin-5-ylmethyl)-D-cysteine (M11a, AppendixIII)

The title compounds were prepared according to the first route depictedin Scheme B hereinabove.

(i) Synthesis of S-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine (M12,Appendix II)

L-cysteine hydrochloride hydrate (37 mg, 0.31 mmol) was dissolved inDMSO (3 ml). To the solution powdered KOH (36 mg, 0.34 mmol) was added,and the mixture was stirred for 30 min at room temperature. Then,powdered 5-chloromethyl-8-hydroxyquinoline hydrochloride (A2) (65 mg,0.34 mmol) was added. The suspension was stirred at room temperature 12h, 2 N HCl was added and the pH was adjusted to 5. The precipitate waswashed with water and acetone. The crude product was further purified bysemi-preparative HPLC to yield compound M12: 53 mg (61%). HPLC (t_(R)):38.1 min (linear gradient: 50% B for the first 4 min, increased linearlyto 100% B for 60 min) [α]_(D) ²⁰=20.5° (c=1.0, H₂O); ¹H NMR (250 MHz,D₂O) 2.89 (d, J=5.6 Hz, 2H), 3.95 (dd, J=5.1, 5.0 Hz, 1H), 4.18 (s, 2H),7.25 (d, J=8.0 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.96 (dd, J=8.7, 5.5 Hz,1H), 8.88 (d, J=5.4 Hz, 1H), 9.22 (d, J=8.7 Hz, 1H); IR (KBr) cm⁻¹: 3438(broad), 3081, 1685, 1637, 1560; Mass spectrometry: calculated forC₁₂H₁₂N₂O₃ m/z [M+H]⁺=279.33. found [M+H]⁺=279.13.

(ii) Synthesis of S-(8-hydroxyquinolin-5-ylmethyl)-D-cysteine (M11,Appendix II)

Compound M11 was prepared as method described in 4(i) above for M12, butusing D-cysteine instead of L-cysteine as starting material. ¹H NMR (250MHz, D₂O) 2.85 (d, J=5.5 Hz, 2H), 3.83 (t, J=5.4, 1H), 4.05 (s, 2H),7.06 (d, J=8.0 Hz, 1H), 7.42 (d, J=8.0 Hz, 1H), 7.86 (dd, J=8.7, 5.4 Hz,1H), 8.77 (d, J=5.3 Hz, 1H), 9.06 (d, J=8.7 Hz, 1H). IR (KBr) cm⁻¹: 3410(broad), 3050, 2926, 1701, 1685, 1550. Mass spectrometry: calculated forC₁₂H₁₂N₂O₃ m/z [M+H]⁺=279.33. found [M+H]⁺=279.26; [α]_(D) ²⁰=−15.3°(c=1.0, H₂O).

(iii) Synthesis of N-propargylS-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine (M12a, Appendix III)

To a stirred mixture of NaHCO₃ (34 mg, 0.4 mmol) and compound M12 (54mg, 0.2 mmol) in 5 ml DMSO, propargyl bromide (24 mg, 0.2 mmol) wasslowly added, and the solution was stirred at room temperature for 24 h.The solvent was removed by vacuum, and the crude product wascrystallized (pH 5.5) and then further purified by semi-preparative HPLC[(C_(H); solvent A=water, 0.1% v/v TFA; solvent B═CH₃CN: water=3: 1,0.1% v/v TFA; t_(R)=24.4 min (linear gradient 0-80% B over 55 min)] togive the title compound M12a: 37 mg, 60% yield. [α]_(D) ²⁰=−16.3.(c=1.0, 0.1 N HCl). Mass spectrometry: calculated for C₁₆H₁₆N₂O₃S m/z[M+H]⁺=317.38. found [M+H]⁺=317.30. ¹H NMR (250 MHz, CHCl₃+D₂O) 2.34 (m,1H), 2.89 (d, J=5.6 Hz, 2H), 3.37 (s, 2H), 3.91 (dd, J=5.1, 5.0 Hz, 1H),4.13 (s, 2H), 7.25 (d, J=8.0 Hz, 1H), 7.57 (d, J=8.0 Hz, 1H), 7.96 (dd,J=8.7, 5.5 Hz, 1H), 8.88 (d, J=5.4 Hz, 1H), 9.22 (d, J=8.7 Hz, 1H); IR(KBr) cm⁻¹: 3428 (broad), 3091, 1695, 1637, 1568.

(iv) Synthesis of N-propargylS-(8-hydroxyquinolin-5-ylmethyl)-D-cysteine (M11a, Appendix III)

The title compound M11a was prepared according to the procedure forM12a, but using M11 instead of M12 as the starting material. Yield 70%.[α]_(D) ²⁰=−18.3. (c=1.0, 0.1 N HCl). Mass spectrometry: calculated forC₁₆H₁₆N₂O₃S m/z [M+H]⁺=317.38. found [M+H]⁺=317.31. ¹H NMR. (250 MHz,CHCl₃+D₂O) 2.39 (m, 1H), 2.85 (d, J=5.6 Hz, 2H), 3.27 (s, 2H), 3.91 (dd,J=5.1, 5.0 Hz, 1H), 4.13 (s, 2H), 7.25 (d, J=8.0 Hz, 1H), 7.57 (d, J=8.0Hz, 1H), 7.96 (dd, J=8.7, 5.5 Hz, 1H), 8.88 (d, J=5.4 Hz, 1H), 9.22 (d,J=8.7 Hz, 1H); IR (KBr) cm⁻¹: 3422 (broad), 3090, 1691, 1639, 1565.

Example 5 Synthesis of N-propargylS-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine (M12a, Appendix III) andN-propargyl S-(8-hydroxyquinolin-5-ylmethyl)-D-cysteine (M11a, AppendixIII)—Method B

The title compounds can be prepared by another method consisting inpreparation of the N-9-fluorenylmethoxycarbonyl (N-Fmoc) derivatives ofthe compounds M11 and M12, removing the protecting Fmoc group andreacting with propargyl bromide.

(i) Synthesis of N-9-Fmoc-S-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine(M11B, Appendix IV)

The crude compound M11 obtained in Example 4(i) (27.5 mg, 0.1 mmol) wasdissolved in 10% NaCO₃ (12 ml). The solution was stirred and the pH wasadjusted to approximately 8 using small portions of 10% NaCO₃. To thesolution, (9-Fluorenylmethyl) succinimidyl carbonate (36.6 mg, 0.11mmol, 1.1 equiv) in dioxane (2 ml) was added dropwise. The reactionmixture was stirred at room temperature for 12 h. The dioxane wasevaporated under reduced pressure at room temperature, and the aqueousphase was extracted with Et₂O (3×30 ml). The aqueous phase was acidifiedto pH 2-3 with 10% KHSO4 and extracted with EtOAc (3×30 ml). Thecombined EtOAc portions were dried over NaSO₄ and concentrated underreduced pressure. The residue was further purified by semi-preparativeHPLC to yield the title compound: 26 mg (50%) HPLC (t_(R): 38.1 min(linear gradient: 50% B for the first 4 min, increased linearly to 100%B 60 min) ¹H NMR (250 MHz, CD₃OD) 2.71 (dd, J=14.1, 9.1 Hz, 1H), 2.95(dd, J=14.2, 4.5 Hz, 1H), 4.07 (t, J=7.1 Hz, 1H), 4.23 (m, 5H), 7.19 (m,5H), 7.54 (m, 3H), 7.68 (m, 3H), 8.78 (d, J=4.9 Hz, 1H), 9.13 (d, J=8.6Hz, 1H); IR (KBr) cm⁻¹: 3398, 3063, 2950, 1702, 1598, 1555; Massspectrometry: calculated for C₂₈H₂₄N₂O₅S m/z [M+H]⁺=501.57. found[M+H]⁺=501.51; [α]_(D) ²⁰=−18.3° (c=1.0, 9:1 MeOH/1.0 aqueous HCl).

(ii) Synthesis of N-9-Fmoc-(8-hydroxyquinolin-5-ylmethyl)-D-cysteine(M12B, Appendix IV)

Compound M12B was prepared by the method for synthesizing compound M11Bas described in step 5(i) above, using the appropriate startingmaterials. ¹H NMR (250 MHz, CD₃OD) 2.71 (dd, J=14.2, 9.1 Hz, 1H), 2.96(dd, J=14.2, 4.4 Hz, 1H), 4.08 (t, J=7.0 Hz, 1H), 4.21 (m, 5H), 7.19 (m,5H), 7.55 (m, 3H), 7.72 (m, 3H), 8.79 (d, J=4.9 Hz, 1H), 9.14 (d, J=8.2Hz, 1H); IR (KBr) cm⁻¹: 3397, 3065, 2952, 1701, 1598, 1554; Massspectrometry: calculated for C₂₈H₂₄N₂O₅S m/z [M+H]⁺=501.57. found[M+H]⁺=501.51; [α]_(D) ²⁰=+17.1° (c=1.0, 9:1 MeOH/1.0 aqueous HCl)

(iii) Synthesis of N-propargylS-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine (M12a, Appendix III)

The title compound is obtained by removal of the Fmoc group from thecompound M12B and reaction thereof with propargyl bromide.

(iv) Synthesis of N-propargylS-(8-hydroxyquinolin-5-ylmethyl)-D-cysteine (M11a, Appendix III)

The title compound is obtained by removal of the Fmoc group from thecompound M11B and reaction thereof with propargyl bromide.

Example 6 Synthesis ofN-(4-propargylpiperazin-1-ylethyl)-2-methyl-3-hydroxy-4-pyridinone(HLA20a, Appendix VI)

The title compound was prepared as described in Scheme D by reaction ofN-(2-chloroethyl)-2-methyl-3-hydroxy-4-pyridinone (H2, Appendix VII)(prepared as described in Bijaya L. Rai, Lotfollah Dekhordi, HichamKhodr, Yi Jin, Zudong Liu, and Robert C. Hider J. Med. Chem. 1998,41,3347-3359) with N-propargylpiperazine (obtained as in step (iii) inExample 2):

To a mixture of H2 (375 mg, 2 mmol) and diisopropylethylamine (0.352 ml,2 mmol, 1 eq) in 6 ml CHCl₃ at 0° C., N-propargylpiperazine (245 mg, 2mmol, 1 eq) was added. The mixture was then stirred for 24 h at roomtemperature. CHCl₃ (10 ml) was added and the solution was washed with 5%NaHCO₃ (3×50 ml), brine (2×50 ml), and then dried over Na₂SO₄. The crudeproduct was purified by FC to give the title compound HLA20a: 337 mg,60% yield. H¹ NMR (250 MHz, CDCl3+D2O), 2.23 (m, 1H), 3.28 (d, J=2.44Hz, 2H), 2.54 (s, 8H), 3.42 (dd, 1.5 Hz 2H), 3.70 (dd, J=3.44, 1.5 Hz2H), 7.18 (m, 1H), 7.78 (d, J=7.59, 1H), 2.60 (s 3H) Mass spectrometry:calculated for C₁₅H₂₁N₃O₂ m/z [M+H]⁺=276.35. found [M+H]⁺=276.30.

Example 7 Synthesis ofN-(N-methyl-propargylaminoethyl))-2-methyl-3-hydroxy-4-pyridinone (M30a,Appendix VI)

The title compound was prepared as described in Scheme D by reaction ofH2 and N-methyl-propargylamine as starting compound instead ofN-propargylpiperazine:

A solution of N-methyl-propargylamine hydrochloride (212 mg, 2 mmole) inCH₂Cl₂ (2 ml) was slowly added to a stirred solution of H2 (375 mg, 2mmol) and diisopropylethylamine (0.352 ml, 2 mmol, 1 eq) in 6 ml CH₂Cl₂at 0° C. The mixture was then stirred overnight at room temperature, andthe solvent was removed in vacuum. The crude solid was redissolved in 10ml of CHCl₃, then washed with 5% NaHCO₃ (3×30 ml), brine (2×20 ml). Theorganic layer was dried over Na₂SO₄ and evaporated in vacuum to give thetitle compound M30a. H¹ NMR (250 MHz, CDCl3+D2O), 2.23 (m, 1H), 3.28 (d,J=2.44 Hz, 2H), 2.33 (s 3H), 3.41 (dd, J=3.44, 1.5 Hz 2H), 3.78 (dd,J=3.44, 1.5 Hz 2H), 7.08 (m, 1H), 7.78 (d, J=7.59, 1H), 2.60 (s, 3H).Mass spectrometry: calculated for C₁₂H₁₆N₂O₂ m/z [M+H]⁺=221.27. found[M+H]⁺=221.30.

Example 8 Synthesis ofN-(4-propargylaminoethoxycarbonyl-piperazin-1-ylmethyl))-2-methyl-3-hydroxy-4-pyridinone(HLA16b, Appendix VI)

The title compound HLA16b was synthesized according to the procedure forM30a in Example 7, but using N-propargylaminoethoxycarbonylpiperazine(H1) instead of N-methylpropargylamine as the starting material. Yield64%. H¹ NMR (250 MHz, CDCl3+D₂O) 2.27 (m, 1H), 3.11 (s 2H), 3.29 (dd,J=10.21, 7.45 Hz, 2H), 4.29 (d, J=11.23, 7.12 Hz, 2H), 2.55 (dd, J=4.73,4.50 Hz, 4H), 2.93 (dd, J=4.96, 4.84 Hz, 4H). 3.45 (dd, J=3.44, 1.5 Hz2H), 3.81 (dd, J=3.44, 1.5 Hz 2H), 7.18 (m, 1H), 7.78 (d, J=7.59, 1H),2.56 (s 3H). Mass spectrometry: calculated for C₁₇H₂₄N₄O₄ m/z[M+H]⁺=349.40. found [M+H]⁺=349.51.

Example 9 Synthesis ofN-propargyl-S-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-L-cysteine.(M12b, Appendix VI) andN-propargyl-S-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-D-cysteine(M11b, Appendix VI)

The title compounds were prepared according to the first route depictedin Scheme D hereinabove by reaction of D1 with cysteine, and furtherreaction of the obtained D2 with propargyl bromide, as follows:

(i) Synthesis of S-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-L-cysteine(L-H3, Appendix VII)

L-cysteine hydrochloride hydrate (37 mg, 0.31 mmol) was dissolved inDMSO (3 ml). To the solution, powdered KOH (36 mg, 0.34 mmol) was addedand the mixture was stirred for 30 min at room temperature. Then, H2 (64mg, 0.34 mmol) in DMSO was added. After 24 hours of stirring at roomtemperature, 2 N HCl was added to the reaction mixture and the pH wasadjusted to 5.5. The precipitate was washed with water and acetone. Thecrude product was further purified by semi-preparative HPLC to yield thetitle compound L-H3: 64 mg (70%). HPLC (t_(R)): 32.1 min (lineargradient: 50% B for the first 4 min, increased linearly to 100% B 60min) [α]_(D) ²⁰-20.5° (c=1.0, H₂O). H¹ NMR (250 MHz, CDCl3+D2O), 3.90(m, 1H), 2.92 (d, 5.0 Hz, 2H), 3.07 (m, 2H), 3.78 (m, 2H), 7.10 (d,J=7.0 Hz, 1H), 7.90 (d, J=8.1 Hz, 1H), 2.50 (s, 3H). Mass spectrometry:calculated for C₁₄H₁₈N₂O₄S m/z [M+H]⁺=273.32. found [M+H]⁺=273.22.

(ii) Synthesis ofN-propargyl-S-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-L-cysteine(M12b, Appendix VI)

A mixture of NaHCO₃ (34 mg, 0.4 mmol) and L-H3 (75 mg, 0.34 mmol) wasdissolved in 5 ml DMSO, and the solution was stirred at room temperaturefor 2 h. To the solution propargyl bromide (24 mg, 0.2 mmol) was slowlyadded, and the solution was stirred at room temperature for 24 h. Thesolvent was removed by vacuum, and the crude product was crystallized inwater (pH 5.5) and further purified by semi-preparative HPLC [(C₁₈;solvent A=water, 0.1% v/v TFA; solvent B═CH₃CN: water=3: 1, 0.1% v/vTEA; t_(R)=20.4 min (linear gradient 0-80% B over 55 min)] to give thetitle compound M12b: 37 mg, 60% yield. [α]_(D) ²⁰=−16.3. ¹H NMR (250MHz, CHCl₃+D₂O) 2.39 (m, 1H), 3.35 (d, J=5.6 Hz, 2H), 4.01 (m, 1H), 2.92(d, 5.0 Hz, 2H), 3.07 (m, 2H), 3.78 (m, 2H), 7.20 (d, J=7.0 Hz, 1H),8.01 (d, J=8.1 Hz, 1H), 2.60 (s, 3H). Mass spectrometry: calculated forC₁₄H₁₈N₂O₄ S m/z [M+H]⁺=311.37. found [M+H]⁺=311.40.

(iii) Synthesis ofS-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-D-cysteine (D-H3, AppendixVII)

The title compound was prepared according to the procedure for L-H3, butusing D-cysteine instead of L-cysteine as the starting material. Yield71%. HPLC (t_(R)): 32.1 min (linear gradient: 50% B for the first 4 min,increased linearly to 100% B 60 min) [α]_(D) ²⁰=+19.5° (c=1.0, H₂O). H¹NMR (250 MHz, CDCl3+D2O), 3.89 (m, 1H), 2.91 (d, 5.0 Hz, 2H), 3.07 (m,2H), 3.78 (m, 2H), 7.10 (d, J=7.0 Hz, 1H), 7.90 (d, J=8.1 Hz, 1H), 2.50(s, 3H). Mass spectrometry: calculated for C₁₄H₁₈N₂O₄S m/z[M+H]⁺=273.32. found [M+H]⁺=273.40

(iv) Synthesis of N-propargylS-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-D-cysteine. (M11b, AppendixVI)

The title compound M11b was synthesized according to the procedure forM12b, but using S-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-D-cysteine(D-H3) instead ofS-(2-methyl-3-hydroxy-4-pyridinon-1-ylethyl)-L-cysteine as the startingcompound. Yield 70%, [α]_(D) ²⁰=+17.3. ¹H NMR (250 MHz, CHCl₃+D₂O): 2.30(m, 1H), 3.40 (d, J=5.6 Hz, 2H), 4.01 (m, 1H), 2.98 (d, 5.0 Hz, 2H),3.07 (m, 2H), 3.78 (m, 2H), 7.20 (d, J=7.0 Hz, 1H), 7.89 (d, J=8.1 Hz,1H), 2.58 (s, 3H). Mass spectrometry: calculated for C₁₄H₁₈N₂O₄S m/z[M+H]⁺=311.37. found [M+H]⁺=311.30.

Example 10 Synthesis of N-propargylglycine hydroxamate (M37, Appendix V)

Propargyl bromide (356.9 mg, 3 mmol) was slowly added to a mixture ofglycine hydroxamate (270 mg, 3 mmol) and diisopropylethylamine (407.1mg, 3.15 mmol) in CHCl₃ (20 ml) at 0° C. The mixture was stirred for 24h at room temperature, and CHCl₃ (50 ml) was added. The solution waswashed with 5% NaHCO₃ (3×50 ml), brine (2×50 ml), and then dried overNa₂SO₄. The solution was filtered and evaporated to dryness to give thetitle compound M37: 192 mg, 50% yield. ¹H NMR (250 MHz, CHCl₃+D₂O) 2.33(m, 1H), 3.33 (d, J=5.6 Hz, 2H), 3.13 (s 2H). Mass spectrometry:calculated for C₅H₈N₂O₂S m/z [M+H]⁺=129.13. found [M+H]⁺=129.20.

Example 11 Synthesis ofN-(4-methylpiperazin-1-ylmethylcarbonyl),N-propargyl glycine hydroxamate(M38, Appendix V)

The title compound was prepared by reaction of N-t-butoxy2-(N-chloroacetyl,N-propargyl)amino-acetamide (H14, Appendix VII) withN-methyl piperazine. Compound H4 was obtained by reaction of N-t-butoxy2-amino-acetamide with chloroacetyl chloride and propargyl bromide, asfollows:

(i) Synthesis of N-t-butoxy2-(N-chloroacetyl-N-propargyl)amino-acetamide (H4)

Propargyl bromide (356.9 mg, 3 mmol) was slowly added to a mixture of2-amino-N-t-butoxy-acetamide (438.6 mg, 3 mmol) anddiisopropylethylamine (407.1 mg, 3.15 mmol) in CHCl₃ (20 ml) at 0° C.The mixture was stirred for 24 h at room temperature, and CHCl₃ (50 ml)was added. The solution was washed with 5% NaHCO₃ (3×50 ml), brine (2×50ml), and then dried over Na₂SO₄. The solution was filtered andevaporated to dryness. The residue was dissolved indiisopropylethylamine (258 mg, 2 mmol) in CH₂Cl₂ (25 ml) at roomtemperature, and chloroacetyl chloride (0.239 mL, 339 mg, 3 mmol) wasslowly added at 0° C. over 30 min. After 2 h of stirring at roomtemperature, the solvent was removed by vacuum. The resulted residue wasdissolved in Et₂OAc (60 ml) and washed with saturated NaHCO₃ (2×50 ml),brine (2×50 ml) and dried over Na₂SO₄ overnight. Evaporation in vacuumgave the title compound H4: 390 mg, 50% yield. ¹H NMR (250 MHz, CHCl₃)2.23 (m, 1H), 3.31 (d, J=5.6 Hz, 2H), 3.13 (s 2H). 4.85 (s 2H), 1.38 (s,9H). Mass spectrometry: calculated for C₁₁H₁₇N₂O₃ m/z [M+H]⁺=261.72.found [M+H]⁺=261.61.

(ii) Synthesis of N-(4-methylpiperazin-1-ylmethylcarbonyl)-N-propargylglycine hydroxamate (M38, Appendix V)

N-methylpiperazine (100 mg, 1 mmol, 1 eq) was added to a stirredsolution of chloroform (5 ml), diisopropylethylamine (0.348 ml, 2 mmol,2 eq) and H4 obtained in step (i) above (261 mg, 1 mmol) at roomtemperature After being stirred for 24 h at room temperature, 10 ml ofCHCl₃ was then added and the solution was washed with 5% NaHCO₃ (3×10ml), brine (2×10 ml), and then dried over Na₂SO₄. The solution wasfiltered and evaporated to dryness. The residue was dissolved in asolution of TFA:H₂O:TES:thioanisole (85:5:5:5) (5 ml), and stirred for 1h at room temperature. After removal of the solvent under vacuum, theresidue was dissolved in EtOAc (50 ml), washed with saturated NaHCO₃(3×20 ml), brine (2×10 ml), and then dried over Na₂SO₄. Evaporation invacuum gave the title compound M38: 160 mg, 60% yield. ¹H NMR (250 MHz,CHCl₃+D₂O) 2.30 (m, 1H), 3.31 (d, J=5.6 Hz, 2H), 3.13 (s 2H), 2.83 (s,3H), 2.44 (s, 8H), 3.87 (s, 2H) Mass spectrometry: calculated forC₁₂H₂₀N₄O₃ m/z [M+H]⁺=269.31. found [M+H]⁺=269.40.

Example 12 Synthesis ofN-[4-(2-hydroxyethyl)piperazin-1-ylmethylcarbonyl), N-propargylglycinehydroxamate (M39, Appendix V)

The title compound M39 was synthesized according to the procedure forM38 in Example 11 above, but using 4-(2-hydroxyethyl)piperazine insteadof N-methylpiperazine as the starting material. Yield 72%. Massspectrometry: calculated for C₁₃H₂₂N₄O₄ m/z [M+H]⁺=299.34. found[M+H]⁺=299.40. ¹H NMR (250 MHz, CHCl₃+D₂O): 2.25 (m, 1H), 3.31 (d, J=5.6Hz, 2H), 3.13 (s 2H), 4.01 (dd, J=5.6, 1.5 Hz, 2H), 3.56 (dd, J=6.6, 1.5Hz, 2H), 2.44 (s, 8H), 3.87 (s, 2H).

Example 13 Synthesis ofN-[4-ethoxycarbonylpiperazin-1-ylmethylcarbonyl), N-propargylglycinehydroxamate (M40, Appendix V)

The title compound M40 was prepared as described in Example 11 above,but using 4-ethoxycarbonylpiperazine instead of N-methylpiperazine asthe starting material. Yield 78%. Mass spectrometry: calculated forC₁₄H₂₂N₄O₅ m/z [M+H]⁺=327.35. found [M+H]⁺=327.28. H¹ NMR (250 MHzCDCl₃+D₂O): 1.28 (dd, J=7.1, 7.1 Hz 3H), 3.81 (s, 2H), 2.44 (s, 4H),3.43 (s, 4H), 4.01 (dd, J=14.21, 7.12 Hz, 2H), 2.33 (m, 1H), 3.33 (d,J=5.6 Hz, 2H), 3.13 (s, 2H).

Example 14 General Peptide Synthesis

The peptides used in the preparation of the compounds of the inventionof formula I such as those depicted in Appendix I, are prepared by thegeneral method as described below.

Unless otherwise stated, all chemicals and reagents were of analyticalgrade. Trifluoroacetic acid (TFA) for high performance liquidchromatography (HPLC) was obtained from Merck (Darmstadt, Germany).N-9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid derivatives andRink amide resins were purchased from Novabiochem (Laufelfingen,Switzerland).

The peptides were prepared manually by solid-phase peptide synthesisusing Fmoc chemistry following the company's protocols. N-α-Fmoc-aminoacid derivatives were used in the synthesis. N-Methyl morpholine (NMM)and benzotriazol-1-yloxy-tris-pyrrolidino-phosphonium hexafluorophsphate(PyBOP) and, when necessary, a combination of1,3-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBO,were utilized as coupling agents. N-methyl pyrrolidone (NMP) and DMFwere used as solvent. Before each coupling, the deprotection of theα-amino group was achieved by reaction with 20% piperidine. Allsynthesized peptides were deprotected and cleaved from the resin using asolution of TFA: triethylsilane (TES): thioanisole:water (85:5:5:5). Thecleavage mixtures were filtered and the peptides were precipitated fromthe solution with peroxide-free dry ether at 0° C. Precipitated peptideswere washed with cold dry ether, dissolved in water orwater/acetonitrile solution, and lyophilized. The crude peptides weresubjected to semipreparative HPLC purification, performed on a Waterssystem composed of two model 510 pumps, model 680 automated gradientcontroller, and model 441 absorbance detector (Waters, Milford, Mass.).The column effluents were monitored by UV absorbance at 214/254 nm. HPLCprepacked columns employed (Merck, Darmstadt, Germany) were LichroCART250-10 mm containing Lichrosorb RP-18 (7 m) for semipreparativepurifications and Lichrospher 100 RP-18, 250-4 mm (5 m) for analyticalseparations. Separations were achieved using gradients of acetonitrilein water containing 0.1% TFA. The solutions containing purified peptideswere lyophilized overnight. Molecular weights of all peptides wereconfirmed by mass spectrometry. Mass spectrometry was performed on aMicromass Platform LCZ4000 (Manchester, UK) utilizing electron sprayionization method. For amino acid composition analysis, peptides werehydrolyzed in 6 N HCl at 100° C. for 24 h under vacuum, and thehydrolyzates were analyzed with a Dionex Automatic Amino Acid Analyzer.

Example 15 Synthesis of VIP analog derivative containing a8-hydroxyquinoline (HQ) group—Fmoc-KKC(HQ)L-NH₂ (M7, Appendix II)

Vasoactive intestinal peptide (VIP) is a 28-mer peptide known to provideneuroprotection against p-amyloid toxicity in models of Alzheimer'sdisease. Mapping of the active site of VIP lead to a peptide of fouramino acids: Lys-Lys-Tyr-Leu. An analog of this VIP fragment wasprepared by replacing the Tyr residue by a Cys residue for linking to a8-hydroxyquinoline (HQ) residue, thus obtaining the compounds of theinvention M7 (Appendix II) and M7A (Appendix I) (see Example 16 below).Although both compounds M7 and M7A were given the same connotationFmoc-KKC(HQ)L-NH₂ in Appendices II and I, respectively, it is clear fromthe structural formula of M7A that it carries a propargylamino grouplinked to the methylene radical at position 5 of the 8-hydroxyquinolinyl(HQ) radical.

To Rink amide resin (71 mg, 33 μmole) was added 2 ml 20% piperidine inNMP. After 5 minute shaking, the solution was drained and the resin waswashed three times with DMF. To the resin was added Fmoc-Leu-OH (47 mg,132 μmole, 4 eq) and PyBOP (69 mg, 132 μmole, 4 eq) in 1 ml DMF, and NMM(29 μl, 264 μmol, 8 eq). The resulting mixture was then shaken for 1 h.The liquid was drained and a second coupling was performed with equalamounts of Fmoc-Leu-OH, PyBOP and NMM. The third coupling was performedvia DCC/HOBt activation: Fmoc-Leu-OH (47 mg, 132 μmole, 4 eq), HOBt (18mg, 132 μmol, 4 eq), and DCC (132 μml 1N DMF, 132 μmol, 4 eq) weredissolved in 1 ml DMF. After 1 hour cooling, the precipitateddicyclohexylurea (DCU) was removed and the solution was added to theresin. After shaking overnight and draining the solution, the resin waswashed (DMFx3, DCMx3). A negative ninhydrin test indicated thecompletion of the coupling reaction. Next three coupling cycles withFmoc-Cys(Mmt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, respectively werecarried out according to the procedure above (both PyBOP and DCC). Atthe completion of all four coupling circles, the resulting resin wastreated with a solution of TFA:TES:DCM (dichloromethane) (1:5:94 v/v) toremove the Mmt (4-methoxytrityl) protecting group.

A solution of 5-chloromethyl-8-hydroxyquinoline (32 mg, 165 μmol, 5 eq)and NMP (17 mg, 20 μl, 5 eq) in DMF/DCM (1 ml) were added to the resinand the mixture was shaken overnight. A negative DTNB(5,5′-dithio-bis-(2-nitrobenzoate) test indicated the completion of thereaction. The resulting HQ-modified peptide was cleaved from the resinusing a solution of TFA: H₂O:TES:thioanisole (85:5:5: 5) andprecipitated with ether. The crude HQ-modified title peptide M7 wasfurther purified by semi-preparative HPLC, as described above. Yield:4.8 mg (5.5 μmol; 16.7%, based on the initial amino acid resin loading).Mass spectrometry: m/z 870.6 (MH+) calculated 870.1. Amino acid analysisafter hydrolysis with 6 M HCl at 110° C. for 22 h: Leu 1.00, Lys 2.16.Cys could not be detected due to its destruction under the acidicconditions of hydrolysis.

Example 16 Synthesis of VIP analog derivative containing a8-hydroxyquinoline (HQ) group and a propargylaminogroup—Fmoc-KKC(HQ)L-NH₂ (M7A, Appendix I) (i) Synthesis of5-(N-tert-butoxycarbonyl,N-propargyl)aminomethyl-8-t-butoxy-quinoline(H5, Appendix VII)

5-Chloromethyl-8-hydroxyquinoline was protected as5-chloromethyl-8-t-butoxy-quinoline, and propargylamine was convertedinto N-(tert-butoxycarbonyl)-propargylamine according to knownprocedures.

N-(tert-Butoxycarbonyl)propargylamine (310 mg, 2 mmol) was added inportions over 30 min to a stirred suspension of NaH (88 mg of 60%dispersion in oil, 2.2 mmol) in DMF (5 ml), under nitrogen. After the H₂gas evolution had ceased, 5-chloromethyl-8-O-t-butyl-quinoline (600 mg,2.4 mmol) in DMF (2 ml) was added dropwise below 30° C. (cold-waterbath). Stirring was continued for 3 h and the reaction mixture waspartitioned between EtOAc (3×30 ml) and H₂O (50 ml). The combined EtOAcsolutions were washed with water, dried over Na₂SO₄ and evaporated to abuff solid which was washed with hexane and vacuum dried, giving thetitle compound: 443 mg (60%), ¹H NMR (250 MHz, CHCl₃) 1.31 (s, 9H), 1.41(s, 9H), 2.29 (m, 1H), 3.27 (s, 2H), 3.91 (s, 2H), 7.25 (d, J=8.0 Hz,1H), 7.57 (d, J=8.0 Hz, 1H), 7.96 (dd, J=8.7, 5.5 Hz, 1H), 8.88 (d,J=5.4 Hz, 1H), 9.22 (d, J=8.7 Hz, 1H); Mass spectrometry: calculated forC₂₂H₂₈N₂O₅ m/z [M+H]⁺=369.47. found [M+H]⁺=369.54.

(ii) Synthesis of 5-(propargylaminobromomethyl)-8-hydroxyquinoline (H6,Appendix VII)

Compound H5 from step (i) above (368 mg, 1 mmol) and N-bromosuccinimide(231 mg, 1.3 mmol) were dissolved in 5 ml of CCl₄. To this solution wasadded benzoyl peroxide (2.5 mg, 0.01 mmol), and the reaction mixture washeated at reflux for 12 h. After reflux, the solution was cooled to 0°C., and the solid precipitate was filtered. The filtrate was washed with1 M aqueous Na₂CO₃, saturated aqueous NaS₂O₃, and brine. The organiclayer was dried (Na₂SO₄), filtered, and evaporated to dryness in vacuum.The residue was dissolved in a solution of TFA:H₂O:TES:thioanisole(85:5:5:5) (5 ml). After 1 h, HPLC indicated complete removal of theprotecting groups. The solution was evaporated to dryness and theresidue was dissolved in water (10 ml) and then basified with Na₂CO₃ (pH11), and extracted with Et₂OAc (3×50 ml). The organic layer was washedwith brine (2×50 ml) and dried over Na₂SO₄ overnight. Evaporation invacuum gave the title compound H6: 218 mg, 75% yield. ¹H NMR (250 MHz,CHCl₃) 2.23 (m, 1H), 3.27 (s, 2H), 5.10 (s, 1H), 7.25 (d, J=8.0 Hz, 1H),7.57 (d, J=8.0 Hz, 1H), 7.96 (dd, J=8.7, 5.5 Hz, 1H), 8.88 (d, J=5.4 Hz,1H), 9.22 (d, J=8.7 Hz, 1H); Mass spectrometry: calculated forC₂₂H₂₈N₂O₅ m/z [M+H]⁺=292.14. found [M+H]⁺=292.04.

(iii) Synthesis of VIP analog derivative containing a 8-hydroxyquinoline(HQ) group and a propargylamino group—M7A (Appendix I, Scheme A)

A solution of H6 of step (ii) above (48 mg, 0.165 mmol, 5 eq) and NMP(17 mg, 0.020 ml, 5 eq) in DMF/CH₂Cl₂ (1 ml) was added to the resin(0.033 mmol, 1 eq) obtained in Example 15 above and the mixture wasshaken overnight. A negative DTNB test indicated the completion of thereaction. The resulting HQ-modified peptide was cleaved from the resinusing a solution of TFA:H₂O:TES:thioanisole (85:5:5:5) and precipitatedwith ether. The crude HQ-modified peptide M7A was further purified bysemi-preparative HPLC (t_(R)): 35.1 min (linear gradient: 50% B for thefirst 4 min, increased linearly to 100% B 60 min). Yield: 6 mg (5.5μmol; 16.7%, based on the initial amino acid resin loading). Massspectrometry: calculated for C₄₉H₆₃N₉O₇S m/z [M+H]⁺=923.68. found m/z[M+H]⁺=923.91. Amino acid analysis after hydrolysis with 6 M HCl at 110°C. for 22 h: Leu 1.00, Lys 2.10. Cys could not be detected due to itsdestruction under the acidic conditions of hydrolysis.

Example 17 Synthesis of VIP analog derivative containing a8-hydroxyquinoline (HQ) group—Stearyl-KKC(HQ)L-NH₂ (M6, Appendix II)

Fmoc-Lys(Boc)-Lys(Boc)-Cys(Mmt)-Leu-[Rink amide resin] 33 μmol wassynthesized as described above in Example 15. After removing the Fmocgroup, the free N-terminal function was coupled with stearic acid (38mg, 132 μmol, 4 eq) according to the procedure described above. Anegative ninhydrin test indicated the completion of the couplingreaction. The peptide was cleaved from the resin using a solution ofTFA:H₂O:TES:thioanisole (85:5:5:5) and precipitated with ether. Asolution of 5-chloromethyl-8-hydroxyquinoline (32 mg, 165 μmol, 5 eq)and NMP (17 mg, 20 μl, 5 eq) in DMF/CH₂Cl₂. (1 ml) was added to thecrude peptide. The mixture was shaken overnight at room temperature. Anegative DTNB test indicated the completion of the reaction. Uponcompletion of the reaction, the solution was drained, and the crudepeptide was further purified by semi-preparative HPLC to yield: 4.6 mg(5.0 μmol; 15.3%, based on the initial amino acid-resin loading). HPLC(t_(R)): 38.1 min (linear gradient: 50% B for the first 4 min, increasedlinearly to 100% B 60 min), t_(R)=47.5 min for St-KKCLNH₂ at the sameconditions. Mass spectrometry: m/z 914.9 (MH+), (calculated 914.3).Amino acid analysis after hydrolysis with 6 M HCl at 110° C. for 22 h:Leu 1.00, Lys 2.21. Cys could not be detected due to its destructionunder the acidic conditions of hydrolysis.

Example 18 Synthesis of VIP analog derivative containing a8-hydroxyquinoline (HQ) group and a propargylaminogroup—Stearyl-KKC(HQ)L-NH₂ (M6A, Appendix I)

To the resin (33 μmol, 1 eq) obtained in Example 17 above was added asolution of 5-(propargylaminobromomethyl)-8-hydroxyquinoline H6 (48 mg,0.165 mmol, 5 eq) and NMP (17 mg, 0.020 ml, 5 eq) in DMF/CH₂Cl₂ (1 ml).The mixture was then shaken overnight. A negative DTNB test indicatedthe completion of the reaction. The resulting HQ-modified peptide wascleaved from the resin using a solution of TFA:H₂O:TES:thioanisole(85:5:5:5) and precipitated with ether. The crude HQ-modified peptideM6A was further purified by semi-preparative HPLC (t_(R)): 40.1 min(linear gradient: 50% B for the first 4 min, increased linearly to 100%B 60 min). Yield: 5.8 mg (6 μmol; 18%, based on the initial amino acidresin loading). Mass spectrometry: calculated for C₅₂H₈₇N₉O₆S m/z[M+H]⁺=967.37. found m/z [M+H]⁺=967.21. Amino acid analysis afterhydrolysis with 6 M HCl at 110° C. for 22 h: Leu 1.00, Lys 2.20. Cyscould not be detected due to its destruction under the acidic conditionsof hydrolysis.

Example 19 Synthesis of Substance P analog derivative containing a8-hydroxyquinoline (HQ) group—[Cys⁷(HQ)]-substance P (M27, Appendix II)

[Cys⁷]-Substance P (9.0 mg, 6.9 μmol) was dissolved inN,N′-dimethylformamide (DMF) (350 μl), and NMM (7.6 μl, 69 μmol, 10equiv) was added. After being stirred for 1 h at room temperature,5-chloromethyl-8-hydroxyquinoline hydrochloride (1.5 mg, 7.6 μmol, 1.1equiv) in 150 μl mixed solvent (DMF:DMSO:CH₃CN 3:3:1) was addeddropwise. The reaction mixture was stirred overnight at roomtemperature. The progress of the reaction was monitored by analyticalHPLC. When necessary, additional 5-chloromethyl-8-hydroxyquinolinehydrochloride was added for completion of the reaction. Upon completionof the reaction, the crude peptide was precipitated with ice-coldtert-butyl methyl ether/petroleum ether, collected by centrifugation,and then purified by semi-preparative HPLC. t_(R)=32.27 min (lineargradient: t B=0% at 1.0 ml/min over 5 min; B=0%-58% at 1.0 ml/min over45 min), (t_(R)=32.28 min for [Cys⁷]-substance P in the sameconditions). Amino acid analysis after hydrolysis with 6 MHCl at 110° C.for 22 h: Lys 1.00, Arg 1.09, Glu 2.03, Gly 1.32, Pro 1.93, Leu 1.01,Phe 0.96, Cys 0.92. Cys was detected usingS-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine as standard reference. Metcould not be detected due to its destruction under the acidic conditionsof hydrolysis. Mass spectrometry: calculated for C₆₇H₁₀₁N₁₉O₁₄S₂ m/z[M+H]⁺=1461.77. found [M+H]⁺=1461.15.

Example 20 Synthesis of Substance P analog derivative containing a8-hydroxyquinoline (HQ) group—[Cys⁸(HQ)]-substance P (M28, Appendix II)

Compound M28 was synthesized by the method described in Example 19 abovefor compound M27 using the appropriate starting materials. The crudepeptide M28 was further purified by semi-preparative HPLC. t_(R)=32.10min (linear gradient: t B=0% at 1.0 ml/min over 5 min; B=0%-58% at 1.0ml/min over 45 min), (t_(R)=32.89 min for [Cys⁸]-substance P in the sameconditions). Mass spectrometry: calculated for C₆₇H₁₀₁N₁₉O₁₄S₂ m/z[M+H]⁺=1461.77. found [M+H]⁺ =1461.17. Amino acid analysis afterhydrolysis with 6 M HCl at 110° C. for 22 h: Lys 1.00, Arg 1.18, Glu1.96, Gly 1.22, Pro 1.77, Leu 0.92, Phe 0.91, Cys 0.96. Cys was detectedusing S-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine as standard reference.Met could not be detected due to its destruction under the acidicconditions of hydrolysis.

Example 21 Synthesis of Substance P analog derivative containing a8-hydroxyquinoline (HQ) group and a propargylaminogroup—[Cys⁷(HQ)]-Substance P (M27A, Appendix I)

To [Cys⁷]-substance P (9.0 mg, 6.9 mmol, 1 eq) in DMF (350 μl) was addedNMM (7.6 μl, 69 μmol, 10 equiv). After being stirred for 1 h at roomtemperature, a solution of5-(propargylamino-bromomethyl)-8-hydroxyquinoline H6 (2.2 mg, 76 μmol,1.1 eq) in 150 μl mixed solvent (DMF: DMSO:CH₃CN 3:3:1) was addeddropwise. The reaction mixture was stirred overnight at roomtemperature. The progress of the reaction was monitored by analyticalHPLC. When necessary, additional5-(propargylamino-bromomethyl)-8-hydroxyquinoline was added forcompletion of the reaction. Upon completion of the reaction, the crudepeptide was precipitated with ice-cold tert-butyl methyl ether/petroleumether, collected by centrifugation, and then purified bysemi-preparative HPLC. t_(R)=35.27 min (linear gradient: t B=0% at 1.0ml/min over 5 min; B=0%-58% at 1.0 ml/min over 45 min), (t_(R)=32.28 minfor [Cys⁷]-substance P in the same conditions). Amino acid analysisafter hydrolysis with 6 MHCl at 110° C. for 22 h: Lys 1.00, Arg 1.10,Glu 2.01, Gly 1.32, Pro 1.93, Leu 1.03, Phe 0.96, Cys 0.99. Cys wasdetected usingS-(8-hydroxyquinolin-5-yl-(propargylamino-methyl)-L-cysteine as standardreference. Met could not be detected due to its destruction under theacidic conditions of hydrolysis. Mass spectrometry: calculated forC₇₀H₁₀₄N₂₀O₁₄S₂ m/z [M+H]⁺=1514.78. found [M+H]⁺=1514.25.

Example 22 Synthesis of Substance P analog derivative containing a8-hydroxyquinoline (HQ) group and a propargylaminogroup—[Cys⁸(HQ)]-Substance P (M28A, Appendix I)

The title compound M28A was synthesized according to the procedure forM27A in Example 21 above, but using [Cys⁸]-substance P instead of[Cys⁷]-substance P as the starting material. Yield 75%. Amino acidanalysis after hydrolysis with 6 MHCl at 110° C. for 22 h: Lys 1.00, Arg1.08, Glu 2.11, Gly 1.22, Pro 1.93, Leu 1.01, Phe 1.04, Cys 0.99. Cyswas detected usingS-(8-hydroxyquinolin-5-yl-(propargylaminomethyl)-L-cysteine as standardreference. Met could not be detected due to its destruction under theacidic conditions of hydrolysis. Mass spectrometry: calculated forC₇₀H₁₀₄N₂₀O₁₄S₂ m/z [M+H]⁺=1514.78. found [M+H]⁺=1514.88.

Example 23 Synthesis of GnRH analog derivative containing a8-hydroxyquinoline (HQ) group—L-Cys⁵(HQ)]GnRH (M8, Appendix II)

Compound M8 was synthesized according to the method for synthesizingcompound M27 described in Example 19 above, using the appropriatestarting materials. The crude peptide M8 was further purified bysemi-preparative HPLC. t_(R)=26.02 min (linear gradient: t B=0% at 1.0ml/min over 5 min; B=0%-75% at 1.0 ml/min over 45 min), (t_(R)=27.54 minfor [Cys⁵]GnRH under the same conditions). Mass spectrometry: calculatedfor C₅₉H₇₈N₁₈O₁₃S m/z [M+H]⁺=1279.43. found [M+H]⁺=1279.86. Amino acidanalysis after hydrolysis with 6 M HCl at 110° C. for 22 h: Arg 1.00,Ser 1.09, Glu 0.92, Gly 2.21, His 0.88, Pro 0.89, Leu 0.89, Cys 0.85.Cys was detected using S-(8-hydroxyquinolin-5-ylmethyl)-L-cysteine asstandard reference.

Example 24 Synthesis of GnRH analog derivative containing a8-hydroxyquinoline (HQ) group—D-Cys⁶(HQ)]GnRH (M22, Appendix II)

Compound M22 was synthesized according to the method for synthesizingcompound M27 described in Example 19 above, using the appropriatestarting materials. Upon completion of the reaction, the crude peptideM22 was precipitated with ice-cold tert-butyl methyl ether (3 ml). Aftercentrifugation, the solid was dissolved in DDW and purified bypreparative HPLC to yield mg (mmol; %). t_(R)=30.82 min (lineargradient: t B=0% at 1.0 ml/min over 5 min; B=0%-58% at 1.0 ml/min over45 min), (t_(R)=32.57 min for [D-Cys⁶]GnRH under the same conditions).Mass spectrometry: calculated for C₆₆H₈₄N₁₈O₁₄S m/z [M+H]⁺=1386.55.found [M+H]⁺=1386.71.

Example 25 Synthesis of GnRH analog derivative containing a8-hydroxyquinoline (HQ) group and a propargylaminogroup—L-Cys⁵(HQ-Pr)]GnRH (MBA, Appendix I)

[Cys⁵]GnRH (7.7 mg, 6.9 mmol) was dissolved in DMF (350 μl), and NMM(7.6 μml, 69 μmol, 10 equiv) was added. After being stirred for 1 h atroom temperature, 5-(propargyl-aminobromomethyl)-8-hydroxyquinoline H6(2.2 mg, 76 mmol, 1.1 eq) in 150 μl mixed solvent (DMF:DMSO:CH₃CN 3:3:1)was added dropwise. The reaction mixture was stirred overnight at roomtemperature. The progress of the reaction was monitored by analyticalHPLC. When necessary, additional H6 was added for completion of thereaction. Upon completion of the reaction, the crude peptide wasprecipitated with ice-cold tert-butyl methyl ether/petroleum ether,collected by centrifugation. The crude peptide M8A was further purifiedby semi-preparative HPLC. t_(R)=30.12 min (linear gradient: t B=0% at1.0 ml/min over 5 min; B=0%-75% at 1.0 ml/min over 45 min), (t_(R)=27.54min for [Cys⁵]GnRH under the same conditions). Yield: 7.2 mg (5.4 mmol;78%). Mass spectrometry: calculated for C₆₂H₈₁N₁₉O₁₃S m/z[M+H]⁺=1332.46. found [M+H]⁺=1332.66. Amino acid analysis afterhydrolysis with 6 M HCl at 110° C. for 22 h: Arg 1.00, Ser 1.09, Glu0.99, Gly 2.21, His 0.98, Pro 0.89, Leu 0.89, Cys 0.85. Cys was detectedusing S-(8-hydroxyquinolin-5-yl-(propargylaminomethyl)-L-cysteine asstandard reference.

Example 26 Synthesis of GnRH analog derivative containing a8-hydroxyquinoline (HQ) group and a propargylaminogroup—D-Cys⁶(HQ-Pr)GnRH (M22A, Appendix I)

The title compound M22A was synthesized according to the procedure forM27A in Example 23 above, but using [D-Cys⁶]GnRH instead of [Cys⁵]GnRHas the starting material. Yield 70%. Mass spectrometry: calculated forC₆₉H₈₇N₂₀O₁₃S m/z [M+H]⁺=1439.56. found [M+H]⁺=1439.66. Amino acidanalysis after hydrolysis with 6 M HCl at 110° C. for 22 h: Arg 1.00,Ser 1.09, Glu 0.99, Tyr 1.03, Gly 1.11, His 0.98, Pro 0.99, Leu 0.89,Cys 0.85. Cys was detected usingS-(8-hydroxyquinolin-5-yl-(propargylaminomethyl)-L-cysteine as standardreference

Example 27 Synthesis of enkephalin analog derivatives containing a8-hydroxyquinoline (HQ) group—YGGC(HQ)L (M18), YGGC(HQ)M (M19),C(HQ)GGFL (M20), C(HQ)GGFM (M21) (Appendix II) (i) General Procedure forthe Synthesis of Compounds M18, M19, M20, M21

To a solution of Cys¹Met⁵-enkephalin or Cys¹Leu⁵-enkephalin (25 mop inDMF (150 μl) was added NMM (26 μl, 250 μmol). After stirring 1 h at roomtemperature, 5-chloromethyl-8-hydroxyquinoline hydrochloride (61 mg, 25μmol) in 250 μl mixed solvent (DMF:DMSO:CH₃CN 3:3:1) was added dropwise.The reaction mixture was stirred overnight at room temperature. Theprogress of the reaction was monitored by analytical HPLC. Whennecessary, additional 5-chloromethyl-8-hydroxyquinoline hydrochloridewas added for completion of the reaction. Upon completion of thereaction, the crude peptide was precipitated with ice-cold tert-butylmethyl ether/petroleum ether, collected by centrifugation, and thenpurified by semipreparative HPLC (linear gradient: t B=0% at 1.0 ml/minover 5 min; B=0%-58% at 1.0 ml/min over 55 min).

(ii) [Cys⁴(HQ)]-Leu⁵-enkephalin (M18, Appendix II) Mass spectrometry:calculated for C₃₂H₄₀N₆O₈S m/z [M+H]⁺=669.76. found [M+H]⁺=669.62(iii) [Cys⁴(HQ)]-Met⁵-enkephalin (M19, Appendix II) HPLC. t_(R)=26.43min (linear gradient: t B=0% at 1.0 ml/min over 5 min; B=0%-58% at 1.0ml/min over 55 min) (t_(R)=23.87 min for [Cys¹]-Leu⁵-enkephalin in thesame conditions). Mass spectrometry: calculated for C₃₂H₄₀N₆O₈S m/z[M+H]⁺=687.80. found [M+H]⁺=687.79.(iv) [Cys¹(HQ)]-Leu⁵-enkephalin (M20, Appendix II) HPLC. t_(R)=29.56 min(linear gradient: t B=0% at 1.0 ml/min over 5 min; B=0%-58% at 1.0ml/min over 55 min) (t_(R)=27.92 min for [Cys¹]-Leu⁵-enkephalin underthe same conditions). Mass spectrometry: calculated for C₃₂H₄₀N₆O₇S m/z[M+H]⁺=653.76. found [M+H]⁺=653.48(v) [Cys¹(HQ)]-Met⁵-enkephalin (M21, Appendix II) Mass spectrometry:calculated for C₃₁H₃₈N₆O₇S₂ m/z [M+H]⁺=671.80. found [M+H]⁺=671.43.

Example 28 Synthesis of enkephalin analog derivatives containing a8-hydroxyquinoline (HQ) group and a propargylamino group—YGGC(HQ)L(M18A), YGGC(HQ)M (M19A), C(HQ)GGFL (M20A), C(HQ)GGFM (M21A) (AppendixI) (i) General Procedure for the Synthesis of Compounds M18A, M19A,M20A, M21A (Appendix I, Scheme C)

The modified enkephalin peptide ([Cys⁴]-Leu⁵-enkephalin,[Cys⁴]-Met⁵-enkephalin, [Cys¹]-Leu⁵-enkephalin, or[Cys¹]-Met⁵-enkephalin) (25 μmol) was dissolved in DMF (150 μl), and NMM(26 μl, 250 μmol) was added. After 1 h of stirring at room temperature,5-(propargylaminobromomethyl)-8-hydroxy-quinoline H6 (25 μmol) in 250 μlmixed solvent (DMF:DMSO:CH₃CN 3:3:1) was added dropwise. The reactionmixture was stirred overnight at room temperature. The progress of thereaction was monitored by analytical HPLC. When necessary, additional H6was added for completion of the reaction. Upon completion of thereaction, the crude peptide was precipitated with ice-cold tert-butylmethyl ether/petroleum ether, collected by centrifugation, and thenpurified by semi-preparative HPLC (linear gradient: t B=0% at 1.0 ml/minover 5 min; B=0%-58% at 1.0 ml/min over 55 min).

(ii) [Cys⁴(propargyl-HQ)]-Leu⁵-enkephalin (M18A, Appendix I) Yield 70%,HPLC. t_(R)=28.43 min (linear gradient: t B=0% at 1.0 ml/min over 5 min;B=0%-58% at 1.0 ml/min over 55 min) (t_(R)=25.87 min for[Cys⁴]-Leu⁵-enkephalin in the same conditions) Mass spectrometry:calculated for C₃₅H₄₃N₇O₈S m/z [M+H]⁺=722.80. found [M+H]⁺=722.62.(iii) [Cys⁴(propargyl-HQ)]-Met⁵-enkephalin (M19A, Appendix I) Yield 60%,HPLC. t_(R)=27.83 min (linear gradient: t B=0% at 1.0 ml/min over 5 min;B=0%-58% at 1.0 ml/min over 55 min) (t_(R)=23.87 min for[Cys⁴]-Met⁵-enkephalin in the same conditions). Mass spectrometry:calculated for C₃₅H₄₃N₇O₈S m/z [M+H]⁺=740.84. found [M+H]⁺=740.70.(iv) [Cys¹(propargyl-HQ)]-Leu⁵-enkephalin (M20A, Appendix I) Yield 72%,HPLC. t_(R)=30.16 min (linear gradient: t B=0% at 1.0 ml/min over 5 min;B=0%-58% at 1.0 ml/min over 55 min)=27.92 min for [Cys¹]-Leu⁵-enkephalinunder the same conditions). Mass spectrometry: calculated forC₃₅H₄₃N₇O₇S m/z [M+H]⁺=706.80. found [M+H]⁺=706.88.(v) [Cys¹(propargyl-HQ)]-Met⁵-enkephalin (M21A, Appendix I) Yield 78%,HPLC. t_(R)=27.26 min (linear gradient: t B=0% at 1.0 ml/min over 5 min;B=0%-58% at 1.0 ml/min over 55 min) (t_(R)=25.90 min for[Cys¹]-Leu⁵-enkephalin under the same conditions). Mass spectrometry:calculated for C₃₄H₄₁N₇O₇S₂ m/z [M+H]⁺=724.84. found [M+H]⁺=724.94.

Example 29 Synthesis of VIP, GnRH, and Substance P analog derivativescontaining a propargylamino group and a hydroxamate group—Compounds M6B,M7B, M8B, M22B, M27B, and M28B (n=1) (Appendix V, Scheme C)

(i) General Procedure for the Synthesis of Hydroxamate Compounds M6B,M7B, M8B, M22B, M27B, and M28B (n=1) (Appendix V)

The modified peptide (7 μmol, 1 equiv.) (St-KKCL-NH₂, Fmoc-KKCL-NH2,[Cys⁵]GnRH, [D-Cys⁶]GnRH, [Cys⁷]-substance P, or [Cys⁸]-substance Psynthesized as described above) was dissolved in DMF (350 μl), and NMM(7.6 μl, 70 μmol, 10 equiv) was added. After 1 hour of stirring at roomtemperature, N-t-butoxy 2-(N-chloroacetyl-N-propargyl)-amino-acetamide(H4) (2 mg, 7.7 μmol, 1.1 equiv) in 150 μl DMF was added dropwise. Themixture was stirred overnight at room temperature. The progress of thereaction was monitored by analytical HPLC. When necessary, additional H4was added for completion of the reaction. Upon completion of thereaction, the solvent was removed under vacuum. To the residue was addeda solution (2 mL) of TFA:H₂O:TES:thioanisole (85:5:5:5). The mixture wasstirred for 1 h and then precipitated with ice-cold tert-butyl methylether/petroleum ether, collected by centrifugation; the crude modifiedpeptide was further purified by semi-preparative HPLC.

(ii) Synthesis of Compound M6B (n=1)(Appendix V)

Yield 71%, HPLC (t_(R)): 25.1 min (linear gradient: 50% B for the first4 min, increased linearly to 100% B 40 min). Mass spectrometry:calculated for C₄₆H₈₅N₉O_(B)S m/z [M+H]⁺=925.29. found m/z [M+H]⁺=925.21Amino acid analysis after hydrolysis with 6 M HCl at 110° C. for 22 h:Leu 1.00, Lys 2.20. Cys could not be detected due to its destructionunder the acidic conditions of hydrolysis.

(iii) Synthesis of Compound M7B (n=1)(Appendix V)

Yield: 68% HPLC (t_(R)): 25.1 min (linear gradient: 50% B for the first4 min, increased linearly to 100% B 50 min. Mass spectrometry:calculated for C₄₃H₆₁N₉O₉S m/z [M+H]⁺=881.07. found m/z [M+H]⁺=881.15.Amino acid analysis after hydrolysis with 6 M HCl at 110° C. for 22 h:Leu 1.00, Lys 2.15. Cys could not be detected due to its destructionunder the acidic conditions of hydrolysis.

(iv) Synthesis of Compound M8B (n=1)(Appendix V)

Yield: 70%. HPLC t_(R)=29.1 min (linear gradient: t B=0% at 1.0 ml/minover 5 min; B=0%-75% at 1.0 ml/min over 45 min), (t_(R)=27.54 min for[Cys⁵]GnRH under the same conditions). Mass spectrometry: calculated forC₅₆H₇₉N₁₉O₁₅S m/z [M+H]⁺=1291.57. found [M+H]⁺=1291.66. Amino acidanalysis after hydrolysis with 6 M HCl at 110° C. for 22 h: Arg 1.00,Ser 1.12, Glu 0.95, Gly 2.16, His 0.98, Pro 0.89, Leu 0.99. Cys couldnot be detected due to its destruction under the acidic conditions ofhydrolysis.

(v) Synthesis of Compounds M22B (n=1)(Appendix V)

Yield 75%. HPLC t_(R)=25.1 min (linear gradient: t B=0% at 1.0 ml/minover 5 min; B=0%-100% at 1.0 ml/min over 45 min), (t_(R)=23.54 min for[D-Cys⁶]-GnRH under the same conditions). Mass spectrometry: calculatedfor C₆₃H₈₅N₁₉O₁₆S m/z [M+H]⁺=1397.53. found [M+H]⁺=1397.61 Amino acidanalysis after hydrolysis with 6 M HCl at 110° C. for 22 h: Arg 1.00,Ser 1.09, Glu 0.99, Tyr 1.03, Gly 1.11, His 0.98, Pro 0.99, Leu 0.89.Cys could not be detected due to its destruction under the acidicconditions of hydrolysis.

(vi) Synthesis of compound M27B (n=1) (Appendix V)

Yield 60%. HPLC. t_(R)=33.27 min (linear gradient: t B=0% at 1.0 ml/minover 5 min; B=0%-58% at 1.0 ml/min over 45 min), (t_(R)=32.28 min for[Cys⁷]-substance P in the same conditions). Amino acid analysis afterhydrolysis with 6 M HCl at 110° C. for 22 h: Lys 1.00, Arg 1.10, Glu2.01, Gly 1.32, Pro 1.93, Leu 1.03, Phe 0.96. Cys and Met could not bedetected due to its destruction under the acidic Conditions ofhydrolysis. Mass spectrometry: calculated for C₆₄H₁₀₂N₂₀O₁₆S₂ m/z[M+H]⁺=1472.75. found [M+H]⁺=1472.55.

(vii). Synthesis of Compound M28B (n=1) (Appendix V)

Yield 65%. HPLC. t_(R)=32.91 min (linear gradient: t B=0% at 1.0 ml/minover 5 min; B=0%-58% at 1.0 ml/min over 45 min), (t_(R)=31.28 min for[Cys⁸]-substance P in the same conditions). Amino acid analysis afterhydrolysis with 6 M HCl at 110° C. for 22 h: Lys 1.00, Arg 1.08, Glu2.11, Gly 1.22, Pro 1.93, Leu 1.01, Phe 1.04, Cys 0.99. Cys and Metcould not be detected due to its destruction under the acidic conditionsof hydrolysis. Mass spectrometry: calculated for C₆₃H₁₀₀N₂₀O₁₆S₂[M+H]⁺=1458.73. found [M+H]⁺=1458.84.

II Biological Section:

Methods (a) Metal Binding Properties

It is known that 8-hydroxyquinoline is a strong chelator for iron andhas a higher selectivity for iron over copper. It is an importantprecondition for the antioxidative-type drugs because it is theexcessive iron stores and iron-mediated generation of free radicals inthe brain that are thought to be associated with neurodegenerativediseases. Therefore, only chelators with a higher selectivity for ironover copper are expected to chelate iron instead of copper and havepotential neuroprotective effects. In order to discuss possiblecorrelation between chelating properties of 8-hydroxyquinoline and itsderivatives with their anti-oxidative ability, and the correlationbetween its derivative and the best established iron chelating drug,desferal, with antioxidative properties, a reliable measurement of thestability constants of the newly synthesized compounds is necessary. Aspectrophotometric method was used for measurement of the iron-complexesstability constants of the compounds.

(b) Partition Coefficients

The partition coefficient (P_(ow)) or the distribution ratio (D_(ow))between octanol and water are most commonly used measures of thehydrophobicity of compounds. D_(ow) can depend on the concentration ofsolute, but at reasonably low concentrations. If the solute is in thesame form in both phases, D_(ow) becomes constant and is called thepartition coefficient P_(ow). P_(ow) plays an important role indetermining the physico-chemical properties and the behaviour ofnumerous organic compounds in biological systems. P_(ow) is applied toquantitative structure-activity relationship studies, and as a measureof hydrophobicity. All the partition measurements are carried out usinga 1-octanol/water shake-flask procedure (Leo, A J. 1991, “Hydrophobicparameter: measurement and calculation”, Methods Enzymol. 202:544-91) orsimply by the elution time of solute from HPLC column. Thereafter, thetransport properties of the iron-complexes are examined in thewater/lipid membrane system (liposomes), which mimics biomembranes muchbetter than the octanol/water system (Breuer et al, 1995).

1-Octanol (Riedel-deHaën, synthesis grade (250-270 nm abs. <0.06) andglass distilled deionized water were used as the partitioning solutions.A Hewlett-Packard 8450A diode array spectrophotometer was used for thequantitative determination of the tested com-pounds in the standardsolutions and in the partitioned solutions. The aqueous phase stocksolutions were shaken with an excess of 1-octanol to presaturate themand were then allow-ed to stand overnight before use. The 1-octanolstock solutions were also presaturated with 10 mM NaOH and allowed tosettle overnight. The experiments were performed in 10-ml stopperedcentrifuge tubes. The tubes were inverted gently for 5 min and then, toassure complete phase separation, they were centrifuged for 20 min at1,000-2,000 g. The aqueous and organic phases were removed separatelyand analyzed by UV spectrophotometry.

(c) Antioxidative Properties

Free radicals and reactive oxygen species generated in biologicalsystems are thought to be responsible, among other factors, forneurodegenerative diseases. The ability of the compounds to reduce therate of formation or decrease the overall yield of free radicals andreactive oxygen species in Fenton and Fenton-type reactions is animportant precondition for the antioxidative-type drugs. Therefore areliable measurement of antioxidative properties of the compounds isnecessary. When the free radical has a long lifetime, direct electronspin resonance (ESR) spectroscopy may be the convenient way to identifythis species. However, for unstable free radicals, like superoxide (O₂

) or hydroxyl radical (.OH) other ways must be used. The influence ofthe compounds on Fenton and Fenton-type reactions is measured bydeoxyribose assay and Spin trapping method.

The deoxyribose assay (Sawahara et al., 1991) is based on thedestruction of deoxyribose by hydroxyl radicals, leading to theformation of malondialdehyde (MDA), which reacts on heating withthiobarbituric acid (TBA), at low pH, to give a pink pigment. Visibleabsorption spectra of this pigment shows very strong and sharpabsorbance with maximum at 532 nm.

The “spin trapping” method has been developed to detect and identify lowconcentration of free radicals in reacting systems. This involves thereaction of a free radical X′ with a diamagnetic compound to form a morestable radical product, which is readily observable by EPR (electronparamagnetic resonance) technique. This method consists of using anitron or nitroso compound to “trap” the initial free radical, as along-lived nitroxide,

(d) Mitochondria Isolation:

Male Sprague-Dawley rats (300-350 g) are decapitated and the brains areimmediately isolated and cooled in ice-cold isotonic 10 mM Tris-HClbuffer (pH 7.5) containing 0.25 M sucrose, 2 mM EDTA and 2% bovine serumalbumin free of fatty acids (isolation buffer), and homogenized with 50ml glass-teflon homogenizer with a motor (Heidolf, Germany) at 200 rpmin a 1:10 (w/v) ratio isolation buffer.

The homogenate is centrifuged at 1000 g for 10 min and the resultantsupernatant then centrifuged at 10,000 g for 10 min. The pellet iswashed with 10 mM Tris-HCl (pH 7.5), 0.25 M sucrose, and centrifugedagain at 10,000 g for 10 min This step is then repeated three moretimes. The pellet is resuspended in 10 mM Tris-HCl (pH 7.5), 0.25 Msucrose at a final concentration of 50-60 mg protein/ml. The samples arestored at −18° C. until use.

(e) Inhibition of Lipid Peroxidation

The ability of the compounds to inhibit lipid peroxidation as initiatedby iron and ascorbate is examined in brain mitochondria preparationemploying the malondialdehyde procedure (Gassen et al. 1996; Ben-Shacharet al., 1991).

The experiments are carried out in triplicates. 7.5 μM of mitochondrialpreparation (0.25 mg protein) are suspended in 750 μM of 25 mM Tris-HCl(pH 7.4) containing 25 μM ascorbic acid. Samples of the drugs to betested are dissolved in water or ethanol and added to the suspension.The reaction is started by the addition of 2.5 or 5 μM FeSO4 (from a 1mM stock solution), and incubation for 2 h at room temperature. Thereaction is stopped by addition of 750 μl of 20% (w/v) trichloroaceticacid. The samples are centrifuged at 12,000 g for 10 mini. 500 μl of thesupernatant is mixed with 500 μl of 0.5% (w/v) thiobarbituric acid andheated t: 95° C. for 30 min. The absorption of thiobarbituric acidderivatives is measured photometrically at X=532 nm. Blank analysis isbased on emission of the mitochondria, or of FeSO4, or alternatively,addition of the drugs after incubation.

(f) Neuroprotective Effects

Neuroprotective effects of the iron chelators are determined both invivo and in vitro systems.

For in vitro experiments, rat pheochromocytoma type 12 (PC12) cells andhuman neuroblastoma SH-SY5Y cells are used to examine theneuroprotective action of the chelators in response to iron andbeta-amyloid toxicity. Cell viability is tested in MTT(2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) testsas well as measuring dopamine and tyrosine hydroxylase by HPLC andrelease of alpha-amyloid (soluble) by Western, since these cells areused as models of dopamine and cholinergic neurons.

The protection in vivo is tested in MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) animal model of PD(Parkinson's disease), a very viable and well-established model ofneurodegeneration, by measuring striatal dopamine and tyrosinehydroxylase, the markers of dopamine neurons.

(g) PC12 Cell Culture.

Rat PC12 cells, originated from rat pheochromocytoma, were grown at 37°C., in a humid 5% CO₂, 95% air environment, in a growth mediumcontaining Dulbecco's modified Eagle's Medium (DMEM, GIBCO, BRL)supplemented with glucose (1 mg/1 ml), 5% fetal calf serum, 10% horseserum, and 1% of a mixture of streptomycin/penicillin. On confluence,the culture was removed and the cells were detached by vigorous washing,centrifuged at 200 g for 5 min and resuspended in DMEM with full serumcontent. 0.5×10⁴ cells/well were placed in microliter plates (96 wells)precoated with collagen (10 μg/cm² were allowed to attach for 24 hbefore treatment).

(h) MTT Tests for Cell Viability.

Twenty-four hours after attachment of the PC12 cells as described in(g), the medium was replaced with DMEM containing 0.1% BSA. The testcompounds were added to the cells after 1 h of incubation. After 24 hincubation, the cells were subjected to MTT test as previously described(Gassen et al., 1998). The absorption was determined in a Perkin-ElmerDual Wavelength Eliza-Reader at λ=570/650 nm after automatic subtractionof background readings. The results are expressed as percentage of theuntreated control.

Example 30 Iron Chelation in Solution

Iron binding was determined by the chelator capacity for restoring thefluorescence of iron-quenched calcein (CAL), a dynamic fluorescentmetallosensor. The iron-scavenging properties of the chelators wereassessed in solution, by mixing iron salts with free CAL.

Iron precomplexed calcein (Fe-CAL) solutions of 1 μM (in Hepes 20 mMbuffered saline, pH 7.4=HBS) were incubated in the presence of theindicated concentrations of different chelators at room temperature andthe fluorescence intensity (475 nm->520 nm) followed with time in aTecan fluorescence plate reader, using 96-well culture plates (Nunc) at100 μl final volume. The time-dependent complexation of iron from Fe-CALby the chelators was followed by fluorescence dequenching. The resultsare shown in FIGS. 1A-1C. The fluorescence intensity attained after 1 hincubation is shown as a function of the chelator concentration.

The corresponding half maximal dequenching level for the correspondingchelator was (in μM) (rank order from the fastest acting to theslowest):

FIG. 1A: DFO (0.5)(not shown)>>HLA16 (3.5)=HLA20 (3.5)>HLM7 (5.0)>HLM8(6.0)>HLM9 (7.2)>L1 (8.3).

FIG. 1B: DFO (0.5)(not shown)>>HLA16 (4.4)>VK-28 (5.2)>HLA20 (5.4)>L1(8.4)>M9 (9.7)>M10 (9.85)

FIG. 1C: DFO (0.5)(not shown)>>VK-28(3.2)>M7 (3.4)>M11 (5.4)>M12 (6)>L1(6.6).

DFO=desferrioxamine B

L1=1,2-dimethyl-3-hydroxy-pyridin-4-one (deferiprone)

Example 31 Iron permeation of the chelators into K562 cells

The iron-scavenging properties of the chelators were assessed in humanerythroleukemia K562 cells, by loading with the permeant CAL-AM probe,in situ formation of free CAL, and binding of cytosolic labile iron.Calcein-AM readily passes through the cell membrane of viable cellsbecause of the enhanced hydrophobicity as compared to Calcein. WhenCalcein-AM permeates into the cytoplasm, it is hydrolyzed by esterasesin cells to the parent Calcein, a pH-independent, cytosolic fluorescentmarker, which remains inside of the cell. The time-dependent recovery offluorescence in the presence of a given chelator provided a continuousmeasure for the capacity of the chelator to access theiron/CAL-containing compartment.

Calcein uptake and intracellular fluorescence were determined asfollows: K562 erythroleukemia cells were preloaded with calcein (CAL) byincubation with 0.25 mM of the fluorescent probe CAL-AM for 5 min at 37°C., washed and incubated in HBS medium containing anti-CAL antibodies inorder to quench traces of extracellular-associated fluorescence. Thesuspensions were placed in 96-well plates (100 μl final volume) andanalyzed as described above while maintaining the system at 37° C. Atthe indicated time, the indicated chelator was added so as to reach afinal 5 μM concentration.

The results are shown in FIGS. 2A-2D. The values of half maximal time(sec) and fraction of fluorescence recovery were:

FIG. 2A: SIH: (125, 1)>HLA20 (175, 1)>HLA16 (225, 0.95)>HLM9 (200,0.50), HLM8 (600, 0.4), L1 (ND)>>DFO (not shown)

FIG. 2B: HLA16 (220, 1)>HLA20 (390, 1)>M10 (1050)>VK-28 (1250,)>>, L1 &M9 (ND)>>>)>>DFO (not shown)

FIG. 2C: SIH: (100, 1)>VK-28 (1500, ?)>>M11, M12, M7 (ND)>>)>>DFO (notshown)

FIG. 2D shows relative permeabilities of the iron chelators HLA20,HLA16, HLM9, VK-28 in K562 cells (values are given relative to thoseobtained with SIH, for which 1.0 represents an apparent rate constant of0.005 sec⁻¹. (equiv. to a t_(1/2) of ingress of 125 sec for a 5 μMchelator solution).

SIH=salicylaldehyde isonicotinoyl hydrazone.

Example 32 Neuroprotection of Differentiated P19 Cells

This experiment was carried out on P19 mouse embryonal carcinoma cellsdifferentiated to neuronal cells.

Differentiated P19 neuronal cells grown in 96-well culture plates weretreated for 6 h with 50 μM 6-OHDA (6-hydroxydopamine) in full mediumcontaining either 5 μM of the indicated chelator (HLM7, HLM8, HLM9,HLA16, HLA20, M7, VK-28, DFO) or 1 μM apomorphine (Apo). The cells weresubsequently washed and resuspended in fresh medium containing 5% Alamarblue, incubated for 4 h, and the fluorescence monitored at 450 nm exc590 nm emission (Tecan Safire fluorescence plate reader). The resultsare shown in FIG. 3. Data are given as % protection, which is equivalentto the % activity relative to the system not treated with 6-OHDA.

Example 33 Neuroprotection of PC12 Cells Against Serum-DeprivationInduced Apoptosis

PC12 cells were grown in serum-free medium alone or in serum-free mediumwith different concentration of the chelators VK-28, M32, HLA20 andRasagiline. After 24 h incubation, the cells were subjected to MTT test.

The results are shown in FIGS. 4A-4B. Cell viability was expressed as apercentage of cells cultured in medium supplemented with serum, whichserved as the control group and was designated as 100%. PC12 cells grownin serum-free medium had a cell viability of 38% (at 24 hr) and wereused as a positive control. Experimental data are shown as mean±SD(r=6).

As shown in FIGS. 4A-4B, the chelators VK-28, HLA20 and M32 allprotected against PC12 cell death after serum withdrawal. After 24 hincubation, the viability of PC12 cells grown in serum-free medium with0.1 μM VK-28, 0.1 μM HLA20, or 0.1 μM M32 was significantly differentfrom that in serum-free medium without the chelators (P<0.01). Thenumber of survival cells was elevated to 80%, 65% and 59%, respectively,as compared with the serum-free control group (38%). At ten or hundredtimes the concentration of the chelators HLA20 (1 or 10 μM) and M32 (1or 10 μM) there was no significant change in the protection against PC12cell death induced by serum withdrawal. With concentration of 1 μM,VK-28 also showed a good inhibitory activity against PC12 apoptosisinduced by serum withdrawal. However, at the concentration of 10 μM, thetoxicity of VK28 became increasingly dominant. This concentration causedabout 75% PC12 cell death in serum-free medium.

Example 34 Inhibitory Effects on MAO Activity in Rat Brain

Monoamine oxidase (MAO), an enzyme that plays a crucial role in themetabolic degradation of biogenic amines, exists in two functionalforms: MAO-A and MAO-B. The two isoenzymes have different substrate andinhibitor specificities.

i. Preparation of Brain MAO

Rats were decapitated and the brains were quickly taken into a weightedice-cold sucrose buffer (0.32 M), and their weights were determined. Allsubsequent procedures were performed at 0-4° C. The brains werehomogenized in 0.25 M sucrose (one part tissue to 20 parts sucrose) in aTeflon glass homogenizer followed by the addition of sucrose buffer to afinal concentration of 10% homogenate. The homogenates were centrifugedat 600 g for 15 min. The supernatant fraction was taken out andcentrifuged at 4500 g for 30 min, the pellet was diluted in 0.32 Msucrose buffer and kept frozen for later assaying of MAO. Proteinconcentration was determined with Bradford reagent at 595 nm, usingbovine serum albumin as a standard.

ii. Determination of MAO Inhibitoy Activity In Vitro

The activity of MAO-A and MAO-B was determined by the adapted method ofTipton & Youdim (1983). The test compound was added to a suitabledilution of the enzyme preparation (70 pg protein for MAO-B and 150 μgMAO-A assay) in 0.05 M phosphate buffer (pH 7.4). The mixture wasincubated together with 0.05 M deprenyl/selegiline, a specific inhibitorof MAO-B (for determination of MAO-A) or 0.05 M clorgylin, a specificinhibitor of MAO-A (for determination of MAO-B). Incubation was carriedfor 1 h at 37° C. before addition of ¹⁴C-5-hydroxytryptamine binoxalate(100 M) for determination of MAO-A, or ¹⁴C-phenylethylamine 100 M fordetermination of MAO-B, and incubation continued for 30 min or 20 min,respectively. The reaction was stopped with 2 M ice-cold citric acid,and the metabolites were extracted and determined byliquid-scintillation counting in cpm units.

iii. Inhibition of MAO-A and MAO-B by the Iron Chelators

MAO-A and MAO-B activities were determined in rat brain homogenate invitro following incubation with varying concentrations of the testcompounds. The inhibitory activities of the tested compounds are shownin FIGS. 5A-5D.

In one experiment, the in vitro inhibitory activity of HLA20, M32,VK-28, M30, M31, and propargylamine (P) was tested against rat brainMAO-B. The test compounds were added to buffer containing 10⁻⁷Mclorgylin and were incubated with the tissue homogenate for 60 min at37° C. before addition of ¹⁴C-β-phenyl-ethylamine. The results are shownin FIGS. 5A-5B. MAO-B activity in presence of the test compound wasexpressed as a percentage of that in control samples. Mean valuesshown±s.e.mean.

In another experiment, the in vitro inhibitory activity of HLA20, M32,VK-28, M30, M31, and propargylamine (P) was tested against rat brainMAO-A. The test compounds were added to buffer containing 10⁻⁷M deprenyland were incubated with the tissue homogenate for 60 min at 37° C.before addition of ¹⁴C-5-hydroxytryptamine. The results are shown inFIGS. 5C-5D. MAO-A activity in presence of the test compound wasexpressed as a percentage of that in control samples. Mean valuesshown±s.e.mean.

These experimental results showed that there were distinct differencesin the inhibitory activities among the test compounds. While ironchelator M30 caused a significant inhibition of both MAO-A and MAO-Bactivities with the IC₅₀ values less than 0.1 μM, chelators M32 and M31had almost no effect on MAO-A and poor inhibitory effect on MAO-Bactivity (17.2% and 14.2% inhibition at 100 μM, respectively). VK-28 andHLA20 showed similar mild inhibitory activities on MAO-A (15.1% and26.1% inhibition at 100 μM, respectively), but on MAO-B activity HLA20exhibited more potent inhibitory effect than that of VK-28 (42.0%inhibition for HLA20 and 13.3% for VK-28). Propargylamine (P)preferentially inhibited both MAO-A and MAO-B activities in aconcentration-dependent manner, with the IC₅₀ values 66.3 μM and 10.1μM, respectively.

Example 35 Transport Property of the Chelators

Lipophilicity plays an important role in drug absorption anddistribution. The logarithm of the n-octanol/water distributioncoefficients (logD) is the most commonly used measure of thelipophilicity of a drug candidate. Here we used an n-octanol/watershake-flask procedure to determine logD. The results (Table 1) indicatedthat the distribution coefficients (logD) of the test chelators variedgreatly with pH values and the compounds tested. In acidic and basicsolution, all the test compounds have low log D values, which refer to agood hydrophilicity. This can be explained by the formation of oxiniumchloride (in acid) and sodium oxinate (in base) derivatives. Table 1shows the order of the lipophilicity of the chelators in water: HLM7(2.19)>HLA16 (1.79)>HLA20 (1.57)>HLM9 (−1.70)>HLM8 (<−2). These datasuggest that HLM9 and HLM8 may have a poor permeability throughbiological membranes due to their highly hydrophilicity; on the otherhand, HLM7 HLA16, and HLA20, because of their lipid solubility, may beable to cross biological membranes.

TABLE 1 Distribution coefficients of the chelators at different pHvalues D^(a) LogD in 0.01N in in 0.01N in 0.01N in in 0.01N CompoundsHCl H2O NaOH HCl H2O NaOH HLA16 0.018 62.96 2.44 −1.74 1.79   0.39 HLM70.043 155.68 0.097 −1.37 2.19 −1.01 HLM8 0.0031 0.0049 0.020 <−2^(c)  <−2^(c)    −1.70 HLM9 0.038^(b) 0.018 0.0014  −1.42^(b) −1.70  <−2^(c)   HLA20 0.025 37.02 0.70 −1.60 1.57 −0.16 ^(a)Distributionratios D = C_(org)/C_(aq) for different compounds, where C_(org). andC_(aq) are concentrations of the measured compound in organic and inaqueous phase respectively. ^(b)D was measured in phosphate buffer (pH =5.64). ^(c)These compounds were found to have Log D values below −2.Since such values are not considered reliable no exact values are given.

Example 36 Inhibition of Lipid Peroxidation in Brain MitochondrialFraction

The radical scavenging/antioxidant properties of the novel chelatorswere determined by the lipid peroxidation assay. This system has beenused to measure the ability of antioxidants to protect biological lipidfrom free radical damage. In the present work, formation of MDA in themitochondria was induced with 50 μM ascorbic acid and 5 μM FeSO₄. Asshown in FIG. 6, all the chelators tested inhibited lipid peroxidation.This inhibition was dose-dependent. The remarkable inhibitory effectswere observed with all the chelators at 25 μM, reducing radical damageby 90% to 97%. At compound concentration as low as 10 μM these chelatorssignificantly reduced radical formation (about 40% inhibition). Thisinhibition may be attributed to radical scavenging and iron chelatingproperties of the test compounds.

Example 37 Topical Photoprotection

In order to determine the level of topical photoprotection provided bythe iron chelators of the invention, guinea pigs are treated topicallywith the test compound, and are then exposed to varying doses of UVradiation to determine the sun protection factor (SPF). Hairless miceare treated topically with the test compound and then subjected tolong-term exposure to a suberythemal dose of UV radiation. The mice areevaluated for skin wrinkling and skin tumors.

In another experiment, guinea pigs are treated topically with the testcompound, sunscreen, and a combination of the two and are then exposedto varying doses of UV radiation to determine the sun protection factor(SPF). Hairless mice are treated topically with the test compound,sunscreen, and a combination of the two and then subjected to long-termexposure to a suberythemal dose of UV radiation. The mice are evaluatedfor skin wrinkling and skin tumors.

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APPENDIX I code name M6A M7A M8A another name StKKC(HQ-Pr)LFmoc-KKC(HQ-Pr)L [L-Cys⁵(HQ-Pr)]GnRH structure

code name M18A M19A another name YGGC(HQ-Pr)L YGGC(HQ-Pr)M structure

code name M20A M21A M22A another name C(HQ-Pr)GGFL C(HQ-Pr)GGFM[D-Cys⁵(HQ-Pr)]GnRH structure

code name M27A M28A structure

APPENDIX II code name M6 M7 M8 another name StKKC(HQ)L Fmoc-KKC(HQ)L[L-Cys⁵(HQ)]GnRH structure

code name M11 M12 M18 another name D-HQ-CysOH L-HQ-CysOH YGGC(HQ)Lstructure

code name M19 M20 M21 another name YGGC(HQ)M C(HQ)GGFL C(HQ)GGFMstructure

code name M22 M27 M28 another name [D-Cys⁶(HQ)]GnRH([Cys⁷(HQ)]-Substance-P [Cys⁸(HQ)]-Substance-P structure

APPENDIX III code name HLA16a HLA20 M9a another name D-(HQ-Pr)-Alastructure

code name M11a M12a M13a another name D-(HQ-Pr)-CysOH L-(HQ-Pr)-CysOHstructure

code name M15a M17 M30 another name structure

code name M31 M33 M34 another name structure

code name M10a another name L-(HQ-Pr)-Ala structure

APPENDIX IV code name VK-28 HLA16 HLM7 another name HQPCOOEt structure

code name HLM8 HLM9 M9 another name HQAla HQAlaEt D-HQ-Ala structure

code name M10 M11B M12B another name L-HQ-Ala structure

code name M13 M15 M32 structure

APPENDIX V code name M6B M7B M8B structure

code name M18B M19B M20B structure

code name M21B M22B M27B structure

code name M28B M35 M36

code name M36a M37 M38 structure

code name M39 M40 M41 structure

code name M42 M43 M44

code name M45 M46 structure

APPENDIX VI code name M9b M11b M12b structure

code name M13b M15b HLA16b structure

code name M17a HLA20a M30a structure

code name M31a M33a M34b structure

APPENDIX VII

H1

H2

H3

H4

H5

H6

1. A method for treatment of a neurodegenerative or cerebrovasculardisease, condition or disorder, which comprises administering to anindividual in need thereof an effective amount of a compound comprisinga moiety having an iron chelator function, said iron chelating moietybeing selected from the group consisting of an 8-hydroxyquinolinemoiety, a hydroxamate moiety, and a pyridinone moiety, and, in addition,one or both of the following moieties: (i) a moiety that imparts aneuroprotective function to the compound, said neuroprotective moietybeing selected from the group consisting of an L- or D-cysteine or an L-or D-alanine residue, a neuroprotective peptide, a neuroprotectivepeptide fragment, and an analog of said neuroprotective peptide orneuroprotective peptide fragment; and (ii) a moiety that impartscombined antiapoptotic and neuroprotective function to the compound,said antiapoptotic and neuroprotective moiety being a propargyl group.2. The method according to claim 1, wherein said antiapoptotic andneuroprotective moiety is a propargylamine group.
 3. The methodaccording to claim 1, wherein said iron chelation function is a moietyof 8-hydroxy-5-quinoline, a 3-hydroxypyridin-4-one or1-hydroxypyridin-2-one of the formulas:

wherein R represents the neuroprotective moiety or the neuroprotectiveand antiapoptotic moiety, and wherein R is linked at position 5, 6 or 7of the quinoline ring, at position 1, 2, 5 or 6 of the3-hydroxy-4-pyridinone, or at position 4 or 5 of the1-hydroxy-2-pyridinone ring, and R′ is C1-C4 lower alkyl.
 4. The methodaccording to claim 1, wherein said compound comprises a8-hydroxy-5-quinolinylmethylene moiety, a2-methyl-3-hydroxy-4-pyridinone, aN-ethylene-2-hydroxy-3-methyl-pyridin-4-one moiety, or a hydroxamatemoity as the iron-chelating function, and as the moiety that imparts aneuroprotective function to the compound, a residue selected from thegroup consisting of a neuroprotective peptide, a neuroprotective peptidefragment, an analog of said neuroprotective peptide or neuroprotectivepeptide fragment, a residue of L- or D-cysteine and a residue L- orD-alanine.
 5. The method according to claim 7 wherein saidneuroprotective peptide is vasoactive intestinal peptide (VIP),gonadotropin-releasing hormone (GnRH), Substance P or enkephalin, andsaid analog of neuroprotective peptide or neuroprotective peptidefragment is an analog of VIP, GnRH, Substance P or enkephalin or of afragment thereof, in which one amino acid residue is replaced by a L- orD-cysteine residue.
 6. The method according to claim 8, wherein saidanalog is selected from the group consisting of the VIP fragment analogsof SEQ ID NO:2 that may bear a stearyl or a Fmoc group at the aminoterminal, the GnRH analogs of SEQ ID NO:4 and SEQ ID NO:5, the SubstanceP analogs of SEQ ID NO:7 and SEQ ID NO:8, and the enkephalin analogs ofSEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
 7. Themethod according to claim 4, further comprising a propargyl group. 8.The method according to claim 7 wherein said hydroxamate is aCONHOH—(CH₂)₂-radical that is linked to the propargyl group via one ormore N atoms or via a piperazine ring.
 9. The method according to claim1, wherein the compound comprises a 8-hydroxy-5-quinolinylmethylenemoiety and a propargyl group, wherein the8-hydroxy-5-quinolinylmethylene moiety is linked to the propargyl groupvia one or more N atoms, via a linker selected from the group consistingof ethylenediamine, piperazine and 1,3,5-perhydrotriazine moiety, or viathe —NH— group of a L- or D-alanine or L- or D-cysteine residue or anester thereof.
 10. The method according to claim 1, wherein saidcompound is a compound of formula I to IV or pharmaceutically acceptablesalt thereof:

wherein R₁ is a residue of an analog of a neuroprotective peptide, or ofa fragment thereof, containing a cysteine residue that is linked to theC atom via the —S— atom of the L- or D-Cys residue, and wherein theamino terminal of the peptide is unsubstituted or substituted by ahydrophobic group; R₂ is H or —NH—X; R₃ is a group selected from thegroup consisting of —NH—CH₂—CH₂—NH—R₄,

—CR₅R₆R₇, —N(CH₃)—X, —N(R₈)—CH(CH₂SH)COOC₂H₅, —N(R₈)—CH₂—COOCH₂C₆H₅, and—S—CH₂—CH(COOH)—NHR₈′; R₄ is a group selected from the group consistingof X, COOC₂H₅, (CH₂)₂—O—R₈; and —COO—(CH₂)₂—NH—R₈; R₅ is H, C₁-C₄ loweralkyl or COOC₂H₅; R₅ is H, COOH, COO⁻ or COOC₂H₅; R₇ is selected fromthe group consisting of —NH—R₈, —NH₃ ⁺, —NH—COCH₃, —NH—NH—R₈, and—NH—NH—CO—CH(CH₂OH)—NH—R₈; R₈ is H or X; R′₈ is H, X or Fmoc; R₉ isselected from the group consisting of H, —CO—CH₂—R₁, —CH₂—COOCH₂C₆H₅,—CH(CH₂SH)COOC₂H₅,

R₁₀ is X; —CH(CH₂SH)COOC₂H₅; or

n is an integer from 1 to 6; R₁₁ is a group selected from the groupconsisting of (i) —S—CH₂—CH(COOH)—NH—X; (ii) —N(X)—CH₂COO—CH₂—C₆H₅;(iii) —N(CH₃)—X; (iv) —N(X)—CH(CH₂SH)COOC₂H₅; (v)—CH₂—NH—NH—CO—CH(CH₂OH)—NH—X; (vi) —C(CH₃) (COOH)—NH—NH—X; (vii)—CH(COOH)—NH—X; (viii) —CH(COOC₂H₅H)—NH—X; and

R₁₂ is X, C₁-C₄ lower alkyl, COOC₂H₅ or —(CH₂)₂—OH—; R₁₃ is X,—CH₂)₂—X—, or —COO—(CH₂)₂—NH—X; and X is a propargyl group.
 11. Themethod according to claim 10, wherein said compound is a compound of theformula I:

or a pharmaceutically acceptable salt thereof wherein R₁ is an analog ofa neuroprotective peptide selected from the group consisting ofvasoactive intestinal peptide (VIP), gonadotropin-releasing hormone(GnRH), Substance P and enkephalin or a fragment thereof, in which oneamino acid residue has been replaced by a L- or D-cysteine residue, saidanalog is linked to the C atom via the —S— atom of the Cys residue, andthe amino terminal of said peptide or fragment thereof is unsubstitutedor substituted by a hydrophobic group; R₂ is H or —NH—X; and X is apropargyl group.
 12. A compound according to claim 11, wherein saidanalog is selected from the group consisting of the analog of VIPfragment of SEQ ID NO:2 bearing a stearyl (identified herein as compoundM6A, Appendix I and compound M6, Appendix II) or a Fmoc group (M7A,Appendix I and M7, Appendix II) at the amino terminal, the residue of aGnRH analog of SEQ ID NO:4 (MBA, Appendix I and M8, Appendix II) or SEQID NO:5 (M22A, Appendix I and M22, Appendix II), the residue of aSubstance P analog of SEQ ID NO:7 (M27A, Appendix I and M27, AppendixII) or SEQ ID NO:8 (M28A, Appendix I and M28, Appendix II), and theresidue of an enkephalin analog of SEQ ID NO:11 (M19A, Appendix I andM19, Appendix II), SEQ ID NO:12 (M21A, Appendix I and M21, Appendix II),SEQ ID NO:13 (M18A, Appendix I and M18, Appendix II), and SEQ ID NO:14(M20A, Appendix I and M20, Appendix II).
 13. The method according toclaim 10, wherein said compound is a compound of the formula II or apharmaceutically acceptable salt thereof:

wherein R₃ is as defined in claim
 10. 14. The method according to claim13, wherein R₃ is: (i) a piperazine ring as represented by the compoundsherein designated HLA16a and M17 (Appendix III), HLA16 (Appendix IV),and HLA20 (Appendix III); (ii) —S—CH₂—CH(COOH)—NHR₈′ and R₈′ is H, asrepresented by the compounds herein designated D-HQ-CysOH (M11, AppendixII) and L-HQ-CysOH (M12, Appendix II), or R₈′ is propargyl, asrepresented by the compounds herein designated D-(HQ-Pr)-CysOH (M11a,Appendix III) and L-(HQ-Pr)-CysOH (M12a, Appendix III), or R₈′ is Fmoc,as represented by the compounds herein designated M11B and M12B(Appendix IV); (iii) —CR₅R₆R₇, wherein R₅ is H, R₅ is COOH, R₇ is—NH—R₈, and R₈ is H, as represented by the compounds herein designatedD-HQ-Ala (M9, Appendix IV) and L-HQ-Ala (M10, Appendix IV); or R₈ ispropargyl, as represented by the compounds herein designatedD-(HQ-Pr)-Ala (M9a, Appendix III) and L-(HQ-Pr)-Ala (M10a, AppendixIII); or R₅ is H, R₆ is COO⁻ and R₇ is —NH₃ ⁺, as represented by thecompound herein designated HQ-Ala (HLM8, Appendix IV); or R₅ is H, R₆ isCOOC₂H₅ and R₇ is —NH₂, as represented by the compound herein designatedHQ-AlaEt (HLM9, Appendix IV); or R₅ and R₆ are both COOC₂H₅, and R₇ is—NH—COCH₃, as represented by the compound herein designated HLM7(Appendix IV); or R₅ is H, R₆ is COOC₂H₅ and R₇ is —NH-propargyl, asrepresented by the compound herein designated M31 (Appendix III); (iv)—NR₈—CH(CH₂SH)COOC₂H₅, wherein R₈ is H, as represented by the compoundherein designated M32 (Appendix IV), or R₆ is propargyl, as representedby the compound herein designated M33 (Appendix III); or (v)—N(CH₃)-propargyl, as represented by the compound herein designated M30(Appendix III).
 15. The method according to claim 10 wherein saidcompound is a compound of formula III or a pharmaceutically acceptablesalt thereof:

wherein R₉ and R₁₀ are as defined in claim
 10. 16. The method accordingto claim 15, wherein R₉ is —CO—CH₂—R₁, wherein R₁ is the residue of ananalog of a neuroprotective peptide or of a fragment thereof containinga L- or D-Cys residue, wherein said analog is selected from the groupconsisting of the residue of an analog of the VIP fragment of SEQ IDNO:2 bearing a stearyl (M6B, Appendix V) or a Fmoc group (M7B, AppendixV) at the amino terminal, the residue of a GnRH analog of SEQ ID NO:4(M8B, Appendix V) or SEQ ID NO:5 (M22B, Appendix V), the residue of aSubstance P analog of SEQ ID NO:7 (M27B, Appendix V) or SEQ ID NO:8(M28B, Appendix V), and the residue of an enkephalin analog of SEQ IDNO:11 (M19B, Appendix V), SEQ ID NO:12 (M21B, Appendix V), SEQ ID NO:13(M18B, Appendix V), and SEQ ID NO:14 (M20B, Appendix V); or the compoundof formula III is represented by the compounds herein designated M35,M36, M37, M38, M39, M40, M41, M42, M43, M44, M45 and M46 (Appendix V).17. The method according to claim 10 wherein said compound is a compoundof formula IV or a pharmaceutically acceptable salt thereof:

wherein R₁₁ is as defined in claim 10, said compound is represented bythe compounds herein designated M9b, M11b, M12b, M13b, M15b, HLA16b,M17a, HLA20a, M30a, M31a, M33a, and M34b (Appendix VI).
 18. The methodaccording to claim 1, wherein said neurodegenerative disease isParkinson's disease or Alzheimer's disease, and said compound is not thecompound 5-[4-(2-hydroxyethyl)piperazin-1-ylmethyl]-8-hydroxyquinoline(designated VK-28 in Appendix IV).
 19. The method according to claim 1,wherein said cerebrovascular disorder is stroke.